Brain Research, 526 (1990) 95-102 Elsevier
95
BRES 15796
Central and peripheral vagal transport of cholecystokinin binding sites occurs in afferent fibers Timothy H. Moran 1, Ralph Norgren:, Robert J. Crosby 1 and Paul R. McHugh 1 1Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (U.S.A.) and eDepartment of Behavioral Science, College of Medicine, Pennsylvania State University, Hershey, PA 17033 (U.S.A.) (Accepted 27 February 1990)
Key words: Cholecystokinin; Receptor; Vagus nerve; Nodose ganglion; Nucleus tractus solitarii; Area postrema
The effects of various vagal lesions on cholecystokinin (CCK) binding sites in the nucleus tractus solitarii (NTS) and area postrema (AP) and the peripheral transport of CCK binding sites in the cervical vagus were examined in rats by in vitro autoradiography with [12sI]CCK-8. Unilateral supraganglionic, but not subdiaphragmatic, vagotomy significantly reduced CCK binding in the ipsilateral NTS. Specific unilateral afferent, but not efferent, vagal rootlet transections also significantly reduced NTS CCK binding ipsilateral to the transections. None of the vagal lesions altered CCK binding in the AP. Infraganglionic but not supraganglionic vagotomy eliminated the peripheral transport of vagal CCK binding sites. Together these results demonstrate that CCK receptors in the NTS are located on vagal afferent terminals, that CCK receptors in the AP are likely postsynaptic to a vagal afferent input and that the peripheral and central transport of vagal CCK binding sites occurs in afferent fibers. INTRODUCTION The presence and axonal transport of cholecystokinin (CCK) binding sites in the cervical 19 and subdiaphragmatic 12 vagus nerve have been demonstrated. CCK binding sites are also present in the nucleus of the solitary tract (NTS) and area postrema (AP) 1°'18, sites of termination for vagal afferent fibers 12. Both of these populations have the pharmacological characteristics of type A CCK receptors 5'9'1°. The localization of type A CCK binding sites in both the NTS and vagus suggests the possibility that the NTS CCK receptors are of vagal origin and that the NTS represents the termination for central transport of CCK binding sites within vagal afferent axons arising from cell bodies in the nodose ganglion. Preliminary data from our laboratory with supraganglionic vagotomy 9 and work by Ladenheim, Speth and Ritter 7 demonstrating a reduction in NTS CCK binding following unilateral nodosectomy support this view. While these binding sites have been autoradiographically identified and pharmacologically characterized, their relationship to each other and to afferent or efferent vagal limbs is not clear. Thus, the present series of experiments were undertaken to characterize the relationship of type A brainstem CCK receptors in the NTS and AP to the transport of vagal CCK binding sites and
tO determine whether the presence and transport of vagal CCK receptors occur in afferent or efferent vagal fibers. In the first experiment, we compared the effect o f unilateral supraganglionic vagotomy outside the posterior lacerated foramen and unilateral subdiaphragmatic vagotomy on CCK binding in the NTS and AP. In the second experiment, we examined the effect of unilateral afferent or efferent vagal rootlet transection on these brainstem CCK receptor populations. We also observed the transganglionic transport of H R P into the NTS and AP after unilateral H R P injections into the cervical vagus of rats subjected to efferent rootlet transection. This permitted an assessment of the contralateral afferent innervation that could be compared with the distribution of NTS and AP CCK receptors after a unilateral afferent rootlet transection. In the final experiment, the effect of unilateral supra- and infraganglionic vagotomy on the cervical transport of CCK binding sites was determined. The logic of this experiment was that both vagotomy procedures disconnect cervical efferent fibers from their cell bodies in t h e dorsal motor nucleus and, if CCK binding sites were present in efferent fibers, both procedures should eliminate C C K binding in distal axons. If CCK binding sites were in afferent" fibers, only the infraganglionic vagotomy disconnects cervical afferent a x o n s from their cell bodies and only this procedure
Correspondence: T.H. Moran, Department of Psychiatry, Meyer 4-119, JohnsHopkins University School of Medicine; Baltimore, MD 21205, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
96 s h o u l d e l i m i n a t e C C K binding. A p r e l i m i n a r y r e p o r t of this w o r k has a p p e a r e d 2.
MATERIALS AND METHODS Male Sprague-Dawley rats served as the subjects for all experiments. Animals were maintained on a 12/12 h light-dark cycle and food was always available unless otherwise specified.
Supra- and infra-ganglionic vagotomies Animals were anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg, i.p.) and placed in a stereotaxic device in a supine position. A midline incision was made and the trachea exposed. The left cervical vagus was exposed and followed by blunt dissection along its course toward the posterior lacerated foramen. The nodose ganglion was identified under 16 power magnification. For supraganglionic vagotomies (n = 8, for central transport, n = 6, for peripheral transport) the ganglion was gently retracted from the foramen and its rostral extent identified. The tips of microscissors were placed along the foramen and the nerve cut above the ganglion. For infraganglionic vagotomies (n = 6), the nerve was severed approximately 0.5 cm distal to the nodose ganglion and retracted ventrally away from the foramen. The wound was closed with surgical clips and rats were returned to their home cages for 3-14 days depending on the particular experiment.
Subdiaphragmatic vagotomies Rats (n = 8) were food-deprived overnight and anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg, i.p.). Following a midline laparotomy, the stomach was placed under gentle retraction and the liver packed off with saline-soaked gauze, exposing the lower part of the thoracic esophagus. Using an operating microscope, the point of branching of the hepatic branch from the right subdiaphragmatic vagus was identified and the trunk proximal and distal to the branch was exposed and stripped of surrounding tissue. TWo ligations of 6-0 silk were placed around the vagal trunk approximately 0.5 cm apart. The areas between the ligations including the hepatic branch was excised and the wound was closed,
Rootlet transections Rats (n = 9) were food-deprived overnight, pretreated with atropine sulfate (0.1 mg/kg i,p.) and then anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg, i.p.). A deep level of anesthesia was maintained with supplemental doses of barbiturate (15 mg/kg) administered approximately every 60 min. Body temperature was monitored continuously and maintained at 37 + 1 °C with a heated surgical stage. A constant pressure respirator was available to relieve any respiratory distress. The surgical procedures were essentially the same as used in a previous publication z5 and will be recounted only briefly here. A detailed methodological paper is in preparation z3. Rats were placed in an atraumatic headholder, positioned supine, and a midline incision made to expose the trachea. After retracting the hyoid bone and the trachea, the base of the skull was exposed deep to the larynx by blunt dissection with the aid of an operating microscope. With the occipital bone cleared of muscle from the level of the bulla caudally to the hypoglossal canal, the jugular foramen was expanded medially with a dental burr and fine ronjeurs to expose the dura mater covering the medulla. Lancing the dura revealed the rootlets of the glossopharyngeal and vagus nerves, as well as the trunk of the accessory nerve. Intracranially, the vagus nerve separates into afferent rootlets, which reach the medulla dorsally, and efferent rootlets, which reach the medulla ventrally. Using fine forceps and iris scissors, the efferent rootlets including the accessory nerve were severed in 3 rats (1 left and 2 right); the afferent rootlets were cut in the 6 other animals (4 left, 2 right). The cranial opening was packed with gelfoam and the wound closed with surgical clips. The rats were then returned to their home cages for 3-10 days.
In order to confirm the rootlet cuts, 7 of the 9 animals were reanesthetized and the cervical vagus exposed on the operated side. Using a micromanipulator, the vagus was raised onto a small polyethylene platform and between 0.8 and 2.0 /~1 of 1.0% horseradish peroxidase conjugated to wheatgerm agglutinin (Sigma) was injected into the nerve via a stout glass pipette (100/~m tip diameter). After this brief (1 h) procedure, the rats were returned to their home cages for an additional 2-3 days, before being sacrificed for the receptor binding assay.
Vagal ligations For experiments addressing the peripheral transport of CCK receptors, 3 days following the supraganglionic and infraganglionic vagotomies, rats were reanesthetized and the cervical vagus exposed. The vagus was stripped of surrounding tissue and ligated with 5-0 silk at least 2 cm below the level of the nodose ganglion. The wound was closed with wound clips and the rats were returned to their home cages. Twenty-four hours later, animals were sacrificed by decapitation and a segment of the ligated nerve was removed. In order to maintain proper orientation, approximately 8 mm of nerve proximal and 4 mm of nerve distal to the ligature were taken. The ligature was removed and the nerve placed in the bottom of a paraffin boat on dry ice and covered with homogenized bovine brain paste. Embedded nerve segments were placed in plastic bags and stored at -70 °C. Ten/~m longitudinal sections were taken through the nerve segments at -15 °C, thaw-mounted onto gelatin-coated slides and dried in a desiccator under partial pressure at -4 °C. Slides were stored at -70 °C until binding procedures were performed.
Sacrifice and tissue sectioning For examining the effect of vagotomies on brainstem CCK receptors, the animals that did not receive HRP injections were sacrificed by decapitation from 5 to 14 days after the vagotomy procedures. The brain was rapidly removed, blocked behind the cortex and the brainstem including the cerebellum rapidly frozen in N-methyl-butane on dry ice. The rats that received vagal HRP injections were anesthetized as above and perfused through the heart with normal saline. Frozen brainstem blocks were placed in plastic bags and stored at -70 °C. Twenty ~m sections were taken through the extent of the NTS and mounted to gelatin-coated slides.
,~
Gelatinous
Subnucleus NTS Commissural NTS I ,',.,--" _,-~-:
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',
"
Posterior NTS
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.... J \ Fig. 1. Schematic representation of coronal caudal brainstem sections. The density of CCK binding was determined at each of the 3 anterior/posterior levels (gelatinous subnucleus, commissural and posterior NTS). Binding densities were compared in the medial and lateral cross hatched areas of the NTS and in the area postrema ipsilateral and contralateral to the vagal lesions. AP, area postrema; SolC, nucleus tractus solitarii, commissural; SolG, nucleus tractus solitarii, gelatinous; SolM, nucleus tractus solitarii, medial; SOIL, nucleus tractus solitarii, lateral; sol, solitary tract.
97 In the non-HRP animals sections were taken in pairs for receptor autoradiography. In HRP-treated animals, sections were taken in triplicate with one set used for HRP histochemistry.
Horseradish peroxidase histochemistry At least 2 slides from each brain were first fixed in 0.05%
glutaraldehyde for 1 h and the processed according to a protocol modified from Mesulam 7 and Gibson et al. 4 to reveal HRP reaction product 12. In this procedure, the sections were immersed serially in the solutions of tetramethyl benzidene and sodium nitroferricyanide for 5 or 8 sequences of 5 min duration. After the reaction process was completed, the sections were cleared in Histoclear, coverslipped
Fig. 2. Autoradiographic depiction of the effects of unilateral subdiaphragmatic (A, B and C) and r i o t supraganglionic vagotomy (D, E and F) at 3 anterior/posterior levels through the NTS and AP in representative animals.
98 and stored at 4 °C. In some preparations, the vagus and nodose ganglion on the operated side were removed and fixed 'en bloc' in 0.05% glutaraldehyde. Once fixed, this tissue was embedded in a gelatin-albuminmixture that was cured rapidly with a few drops of 10% glutaraldehyde. These blocks were then frozen, sectioned at 40 /~m, mounted on slides, and then processed following the same protocol used for the brain sections. Autoradiographic analysis of brainstem binding density was only carried out on rats whose rootlet lesions were substantiated by HRP transport.
AREA POSTREMA 10000
The slide-mounted tissue sections were incubated as previously described9-11 in buffer containing 50 pM [12sI]CCK-8(New England Nuclear). Alternate slides containing adjacent sections were incu-
Subdiaphragmatic Vagotomy ~l|nranannllnnlP. Vaootomu
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Fig. 4. Densities of [125I]CCK binding in the area postrema in response to unilateral subdiaphragmatic or supraganglionic vagotomies. Binding in the AP was not affected by either procedure.
bated in the presence of an excess (1 /~M) unlabelled CCK-8 to determine the extent of non-specific binding. Slides were washed with cold (4 °C) buffer and dried. Dried slides were placed in X-ray cassettes and oppossed to [3H]Ultrofilm (LKB) along with [141]standards (Amersham). Binding under the conditions used has been shown to be saturable, of high affinity, reversible, and characterized by a high ratio of specific to non-specific binding (85-90%). Autoradiographic images from the developed films were directly compared with the corresponding tissue sections, which had been stained with Cresyl violet, by viewing the superimposed autoradiograph and histologic section. This allowed accurate localization of CCK binding sites. Images were analyzed utilizing a computerized microdensitometry system (RAS-1000 Amersham). Specific binding was calculated as the difference in standard generated dpm's between adjacent sections incubated with and without 1 /~M unlabelled CCK-8.
Data analysis
2O00
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1000
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In the experiments examining the origin of NTS and AP CCK receptors, CCK binding density in the NTS was examined at 3 rostral caudal levels. At each level the NTS was divided into 4 quadrants and at the middle level, the AP was divided into 4 quadrants. These are demonstrated in Fig. 1. Data were analyzed by a mixed model ANOVA for the factors of vagotomy type and measurement side comparing regions ipsilateral and contralateral to the vagotomy procedures. A similar design was used for analyzing binding within AP. The AP was divided and the binding densities ipsilateral and contralaterai to the lesions were compared. Binding data for vagal segments were analyzed by ANOVA comparing the binding densities proximal and distal to the ligation for the two vagotomy procedures.
2000
RESULTS 1000
0
Lateral
Medial
CONTRALATERAL
Medial
Lateral
IPSILATERAL
Fig. 3. Densities of [1251]CCK binding in the lateral and medial aspects of the NTS at 3 anterior/posterior levels in response to unilateral subdiaphragmatic or supraganglionic vagotomies expressed as computer-generated DPMs/mg of tissue as a measure of binding density. Stars indicate significant depletions relative to contralateral levels. Supraganglionic but not subdiaphragmatic vagotomies affected binding densities.
Analysis of autoradiographs from rats receiving unilateral supraganglionic or unilateral subdiaphragmatic vagotomies revealed different patterns of [125I]CCK binding depending upon the type of vagotomy and the level of the NTS examined (Fig. 2). In the posterior NTS, supraganglionic vagotomy produced significant decreases in binding density ipsilateral to the lesion in both the medial and lateral aspects of the commissural NTS, expressed as significant interactions on the A N O V A
99 GELATINOUS SUBNUCLEUS 4OOO
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Lateral
IPSILATERAL
Fig. 5. Densities of [1251]CCK binding in the lateral and medial aspects of the NTS at 3 anterior/posteriorlevels followingunilateral efferent or afferent vagal rootlet transections. Stars indicate significant depletions relative to the contralateral levels. Afferent but not efferent rootlet transectionsproduce ipsilateral depletions in binding densities.
(lateral: El,14 = 35.308, P < 0.001; medial El,14 = 8.600, P < 0.01) (Fig. 3). At the level of the commissural NTS, supraganglionic vagotomy decreased binding density only in the lateral aspect ipsilateral to the lesion F1,14 = 6.21, P < 0.05. Supraganglionic vagotomy reduced binding density in the NTS at the level of the gelatinous subnucleus in both the ipsilateral medial aspect corresponding to the gelatinous and medial subnuclei (medial aspect: Fl,14 = 34.396, P < 0.001) and in the lateral aspect corresponding to the lateral subnuclei (lateral aspect: F1.14 = 5.614, P < 0.05). No changes in binding density were evident ipsilateral to the lesion in animals
with subdiaphragmatic vagotomies (Fig. 3). As well, no changes in binding density were found ipsilateral to the lesions in the AP in either group (Fig. 4). Unilateral efferent and afferent vagal rootlet transections differentially affected NTS CCK binding. HRP verification of the completeness of the lesions demonstrated that one animal with an efferent transection also had an afferent cut; in one afferent cut, the transection was incomplete in that there was significant label in the NTS. Data from these animals were not included in the analyses of binding density. Unilateral efferent transections had no effect on CCK binding at any level in either the medial or lateral aspects of the NTS (Fig. 5). In contrast, unilateral afferent vagal lesions produced significant decreases in CCK binding ipsilateral to the lesion at all levels of the NTS. ANOVA demonstrated a significant main effect of lesion in both the lateral and medial aspects of the NTS on the ipsilateral compared to contralateral side: lateral, F1,4 = 15.282, P < 0.02; medial, F1,4 = 7.888, P < 0.05. Planned t-analyses demonstrated significant depletions in binding density in the medial and lateral portions at all three levels of the NTS (Fig. 5). Neither efferent nor afferent rootlet transections produced ipsilateral depletions in the AP. A comparison of the unilateral afferent vagal input to the NTS as demonstrated by HRP transport in rats with unilateral efferent transections and the distribution of NTS CCK receptors in rats with unilateral afferent transections is demonstrated in Fig. 6. HRP verification of the lesion in rats receiving efferent transections demonstrated that unilateral afferent vagal inputs have both ipsilateral and contralateral projections in the NTS. The reaction product in NTS resulting from an HRP injection into the left cervical vagus overlaps substantially with the CCK receptor distribution remaining after a right vagal afferent rootlet transection (compare Fig. 6A, B and C with Fig. 6D, E and F, respectively). Thus, after a unilateral afferent rootlet transection, the high affinity CCK binding is confined to those areas of the NTS that are innervated by the intact, contralateral vagal afferent axons. Analysis of the effects of supra- and infraganglionic vagotomy on the transport of CCK binding sites in the cervical vagus revealed differential effects of the vagotomy procedures proximal but not distal to the vagal ligations, F1,10 = 5.273, P < 0.05. As demonstrated in Fig. 7, following supraganglionic vagotomy, vagal transport of CCK binding sites is intact as indicated by a significant increase in CCK binding proximal to the vagal ligature. This increase in binding density proximal to the ligature was eliminated by the infraganglionic vagotomy procedure.
100
Fig. 6. Comparison of unilateral vagal afferent input (A, B and C) as demonstrated by transganglionic HRP transport from the cervical vagus following a unilateral efferent rootlet transection and [125I]CCKbinding in a rat with a unilateral afferent rootlet transection (D, E and F) at 3 anterior/posterior levels in the NTS. The distribution of CCK binding sites following a unilateral afferent transection overlays the pattern of unilateral afferent vagal input.
DISCUSSION The results of these experiments demonstrate a number of phenomena. First, unilateral supraganglionic vagotomy produces a significant ipsilateral reduction in [125I]CCK binding in the NTS. This result is specific to severing the vagus above the level of the afferent cell bodies in the nodose ganglion as subdiaphragmatic
vagotomy does not alter CCK binding in the NTS. The specific vagal component underlying this transport is demonstrated by the finding that unilateral afferent rather than efferent rootlet transection results in unilateral reductions in CCK binding throughout the NTS. Together these findings demonstrate that a significant portion of CCK binding sites in the NTS are located presynaptically on vagal afferent terminals.
101 Vagal CCK Binding at Cervical L i g a t i o n Following Vagotomy 3000
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2000
•
Proximalto Ligation
•
Distal to Ltgatlon
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Infragangllonic
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Fig. 7. Densitiesof [125I]CCKbindingproximal and distal to ligation of the cervical vagus in rats with either supraganglionic or infraganglioni¢ vagotomies. Significant levels of binding were present proximal to the ligationin rats with supraganglionic,but not infraganglionic, vagotomies. Furthermore, vagal transport of CCK binding sites toward peripheral axon terminals also occurs in afferent fibers because severing the vagus below the nodose ganglion, disconnecting axons from both efferent and afferent cell bodies interrupts this transport while severing the vagus above the nodose, disconnecting axons from only the efferent cell bodies does not. This finding, in combination with the central results demonstrates that the transport of CCK binding sites is bidirectional from afferent cell bodies in the nodose ganglion. In contrast, CCK binding in the area postrema was not affected by any of the vagotomy procedures. This result supports the view that binding sites in the AP are not of vagal origin. Although unilateral supraganglionic vagotomy caused a significant ipsilateral reduction in CCK binding in the NTS, this was a reduction rather than a unilateral elimination. This initial result complements that of Ladenheim, Speth and Ritter 7 with nodosectomy and the lack of a complete elimination may be interpreted in a number of ways. First, it is possible that a significant potion of CCK binding in the NTS is postsynaptic to vagal afferent axons and these sites are not affected by the vagotomy procedure. Alternatively, the extent of the elimination of NTS vagal afferent input produced by either lesion may not be complete. For example, the nodose ganglion does not have a sharp border. Afferent vagal cell bodies stream centrally away from the ganglion ] and either the supraganglionic vagotomy or the nodosectomy may leave a significant portion of afferent fibers connected to their cell bodies. Third, the remaining opposite intact unilateral input may project bilaterally in the NTS. The result of the afferent rootlet transection support the latter two interpretations. Following unilateral after-
ent rootlet transection the magnitude of ipsilateral depletion in CCK binding is somewhat greater than that achieved with supraganglionic vagotomy in that significant ipsilateral depletions are found at all levels of the NTS. This suggests that a greater number of afferent fibers may have been disconnected from their cell bodies with the rootlet transection procedure. In addition, as demonstrated by the HRP injections after efferent rootlet transections, the unilateral afferent vagal input into the NTS has a significant contralateral component. Furthermore, after a unilateral afferent transection, the distribution of NTS CCK receptors demonstrated a close correspondence to the pattern of HRP reaction product produced after labelling intact vagal afferent axons. Thus, although the question of whether all the CCK binding sites in the NTS are presynaptic and of vagal origin cannot be definitively answered from the present data, the patterns of depletion and afferent vagal innervation suggest that this may he the case. While supraganglionic vagotomy or afferent vagal transections affected [125I]CCK binding in the NTS, there were no unilateral reductions in binding density in the AP in response to these lesions. This is a similar result to that demonstrated by Ladenheim, Speth and Ritter 7 in response to unilateral nodosectomy. This result could be interpreted as indicating either that the vagal afferent input into the AP is completely bilateral or that these receptors are not on vagal afferent axons. The bilateral distribution interpretation is not supported by the data. This would imply that, after a unilateral transection the receptor density in the AP would be reduced by 50%. In fact, none of our experimental manipulations altered the density of CCK binding sites in the AP. Thus it appears that CCK binding sites in the AP are not of vagal origin. In as much as the AP is a circumventricular organ with a permeable blood brain barrier, the role of these receptors may be to respond to circulating CCK. The peripheral vagal transport of CCK receptors has been previously demonstrated in both the cervical vagus 19 and in all the subdiaphragmatic vagal branches 11. The movement of the receptors is by fast axonal transport in that it is blocked by colchicine ]9. Previous work, however, had not discerned whether transport occurred in the afferent or efferent limb of the vagus. In the present study, infraganglionic rather than supraganglionic vagotomy prevented the peripheral transport of CCK receptors in the cervical vagus as measured by an accumulation of CCK binding sites at a vagal ligation placed distal to vagal transection. Cell bodies for afferent vagal fibers are contained in the nodose ganglion while cell bodies for efferent vagal fibers are contained within the dorsal motor nucleus of the caudal hindbrain. Since both vagotomy procedures disconnect efferent vagal
102 fibers at the site of the ligation from their cell bodies, both would be expected to eliminate the peripheral transport of vagal C C K receptors if this were occurring in efferent vagal fibers. This was not the case. Only the infraganglionic vagotomy, which disconnected both afferent and efferent vagal fibers at the ligation point from their cell bodies, eliminated vagal C C K receptor transport. Thus this transport of C C K receptors occurs on vagal afferent fibers. The demonstration that vagal CCK receptors are contained in afferent rather than efferent fibers has important implications for the identification of potential sites of action for the feeding inhibitory action of CCK. Type A receptors are likely to mediate the feeding effects of CCK, but the particular receptor population at which this inhibitory action of C C K is mediated remains unclear. Candidate type A receptor populations have been identified in the circular muscle layer of the pyloric sphincter 17, as well as in the vagus and brain stem as discussed here. A role for the afferent vagus in the inhibition of food intake by CCK is evident from results demonstrating that total vagotomy or combined unilateral afferent rootlet transection and contralateral subdia-
phragmatic vagotomy eliminate C C K satiety aS. The present demonstration that it is the afferent vagal fibers which contain C C K receptors suggests that some aspect of CCK's feeding inhibitory action could be through the direct stimulation of these vagal C C K receptors. Circulating C C K may directly activate C C K receptors in the A P and on afferent vagal terminals 14. Circulating CCK is unlikely to have access to C C K receptors of vagal origin in the NTS. These receptors may play some role in modulating vagal afferent input in response to locally released CCK. Such a role is suggested in the work of Ewart and Wingate 3 demonstrating augmentation of NTS responses to gastric distention by iontophoretically applied CCK. Thus C C K receptors are present along the afferent vagus and at its central terminations but how actions of these various C C K receptor populations may interact in the inhibition of food intake by C C K remains to be determined.
REFERENCES
Cholecystokinin Antagonists, Alan R. Liss, New York, 1988, pp. 117-132. 10 Moran, T.H., Robinson, P.H., Goldrich, M.S. and McHugh, P.R., Two brain CCK receptors: implications for behavioral actions, Brain Research, 362 (1986) 175-179. 11 Moran, T.H., Smith, G.P., Hostetler, A.M. and McHugh, P.R., Transport of cholecystokinin (CCK) binding sites in subdiaphragmatic vagal branches, Brain Research, 415 (1987) 149-152. 12 Norgren, R. and Smith, G.P., Central distribution of subdiaphragmatic vagal branches in the rat, J. Comp. Neurol., 237 (1988) 207-223. 13 Norgren, R. and Smith, G.P., A method for selective section of vagal afferent or efferent axons in the rat, in preparation. 14 Raybould, H.E., Gayton, R.J. and Dockray, G.J., CNS effects of circulating CCK-8: involvement of brainstem neurons responding to gastric distention, Brain Research, 342 (1985) 187-190. 15 Smith, G.P., Jerome, C. and Norgren, R., Afferent axons of the abdominal vagus mediate the satiety effects of cholecystokinin in rats, Am. J. Physiol., 249 (1986) R638-R641. 16 Smith, G.P., Jerome, C. Cushin, B.J., Eterno, R. and Simansky, K.J., Abdominal vagotomy blocks the satiety effect of cholecystokinin in the rat, Science, 213 (1981) 1036-1037. 17 Smith, G.T., Moran, T.H., Coyle, J.T., Kuhar, M.J., O'Donahue, T.L. and McHugh, P.R., Anatomical localization of cholecystokinin receptors to the pyloric sphincter, Am. J. Physiol., 246 (1984) R127-130. 18 Zarbin, M.A., Innis, R.B., Wamsley, J.K., Snyder, S.H. and Kuhar, M.J., Autoradiographic localization of cholecystokinin receptors in rodent brain, J. Neurosci., 3 (1983) 877-906. 19 Zarbin, M.A., Wamsley, J.K., Innis, R.B. and Kuhar, M.J., Cholecystokinin receptors: presence and axonal flow in the rat vagus nerve, Life Sci., 29 (1981) 697-705.
1 Aitschuler, S.M., Xinman, B., Bieger, D., Hopkins, D.A. and Miselis, R.M., Viscerotopic representation of the upper alimentary tract in the rat: sensory ganglia and nuclei of the solitary and spinal trigeminal tracts, J. Comp. Neurol., 283 (1989) 248-268. 2 Crosby, R.J., Norgren, R., Moran, T.H. and McHugh, P.R., Central and peripheral transport of CCK-A receptors on vagal afferent fibers, FASEB J., 3 (1989) A998. 3 Ewart, W.R. and Wingate, D.L., Cholecystokinin octapeptide and gastric mechanoreceptor activity in rat brain, Am. J. Physiol., 244 (1983) G613-G617. 4 Gibson, A., Hansma, D., Houk, J. and Robinson, E, A sensitive low artifact TMB procedure for the demonstration of WGA-HRP in the CNS, Brain Research, 298 (1984) 235-241. 5 Hill, D.R., Campbell, N.J., Shaw, T.M. and Woodruff, G.N., Autoradiographic localization and biochemical characterization of peripheral type CCK receptors in the rat CNS using highly specific nonpeptide CCK antagonists, J. Neurosci., 7 (1987) 2967-2976. 6 Kalia, M. and Sullivan, J.M., Brain stem projections of sensory and motor components of the vagus nerve, J. Comp. Neurol., 211 (1982) 248-264. 7 Ladenheim, E.E., Speth, R.C. and Ritter, R.C., Reduction of CCK-8 binding in the nucleus of the solitary tract in unilaterally nodosectomized rats, Brain Research, 474 (1988) 125-129. 8 Mesulam, M.M., Tetramethylbenzidene for horseradish peroxidase neurochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing afferent and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 9 Moran, T.H. and McHugh, P.R., Anatomical and pharmacological differentiation of pyloric, vagal and brainstem cholecystokinin receptors. In R.Y. Wang and R. Schoenfeld (Eds.),
Acknowledgements. This work was supported by NIH Grants DK 19302 and DC 00240 and generous gifts from Mrs. Samuel Hecht and the Lorraine and Leonard Levin Fund for Research in Biological Psychiatry. R.N. is a recipient of an NIMH Research Scientist Award, Level If (MH 00653).
95
BRES 15796
Central and peripheral vagal transport of cholecystokinin binding sites occurs in afferent fibers Timothy H. Moran 1, Ralph Norgren:, Robert J. Crosby 1 and Paul R. McHugh 1 1Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (U.S.A.) and eDepartment of Behavioral Science, College of Medicine, Pennsylvania State University, Hershey, PA 17033 (U.S.A.) (Accepted 27 February 1990)
Key words: Cholecystokinin; Receptor; Vagus nerve; Nodose ganglion; Nucleus tractus solitarii; Area postrema
The effects of various vagal lesions on cholecystokinin (CCK) binding sites in the nucleus tractus solitarii (NTS) and area postrema (AP) and the peripheral transport of CCK binding sites in the cervical vagus were examined in rats by in vitro autoradiography with [12sI]CCK-8. Unilateral supraganglionic, but not subdiaphragmatic, vagotomy significantly reduced CCK binding in the ipsilateral NTS. Specific unilateral afferent, but not efferent, vagal rootlet transections also significantly reduced NTS CCK binding ipsilateral to the transections. None of the vagal lesions altered CCK binding in the AP. Infraganglionic but not supraganglionic vagotomy eliminated the peripheral transport of vagal CCK binding sites. Together these results demonstrate that CCK receptors in the NTS are located on vagal afferent terminals, that CCK receptors in the AP are likely postsynaptic to a vagal afferent input and that the peripheral and central transport of vagal CCK binding sites occurs in afferent fibers. INTRODUCTION The presence and axonal transport of cholecystokinin (CCK) binding sites in the cervical 19 and subdiaphragmatic 12 vagus nerve have been demonstrated. CCK binding sites are also present in the nucleus of the solitary tract (NTS) and area postrema (AP) 1°'18, sites of termination for vagal afferent fibers 12. Both of these populations have the pharmacological characteristics of type A CCK receptors 5'9'1°. The localization of type A CCK binding sites in both the NTS and vagus suggests the possibility that the NTS CCK receptors are of vagal origin and that the NTS represents the termination for central transport of CCK binding sites within vagal afferent axons arising from cell bodies in the nodose ganglion. Preliminary data from our laboratory with supraganglionic vagotomy 9 and work by Ladenheim, Speth and Ritter 7 demonstrating a reduction in NTS CCK binding following unilateral nodosectomy support this view. While these binding sites have been autoradiographically identified and pharmacologically characterized, their relationship to each other and to afferent or efferent vagal limbs is not clear. Thus, the present series of experiments were undertaken to characterize the relationship of type A brainstem CCK receptors in the NTS and AP to the transport of vagal CCK binding sites and
tO determine whether the presence and transport of vagal CCK receptors occur in afferent or efferent vagal fibers. In the first experiment, we compared the effect o f unilateral supraganglionic vagotomy outside the posterior lacerated foramen and unilateral subdiaphragmatic vagotomy on CCK binding in the NTS and AP. In the second experiment, we examined the effect of unilateral afferent or efferent vagal rootlet transection on these brainstem CCK receptor populations. We also observed the transganglionic transport of H R P into the NTS and AP after unilateral H R P injections into the cervical vagus of rats subjected to efferent rootlet transection. This permitted an assessment of the contralateral afferent innervation that could be compared with the distribution of NTS and AP CCK receptors after a unilateral afferent rootlet transection. In the final experiment, the effect of unilateral supra- and infraganglionic vagotomy on the cervical transport of CCK binding sites was determined. The logic of this experiment was that both vagotomy procedures disconnect cervical efferent fibers from their cell bodies in t h e dorsal motor nucleus and, if CCK binding sites were present in efferent fibers, both procedures should eliminate C C K binding in distal axons. If CCK binding sites were in afferent" fibers, only the infraganglionic vagotomy disconnects cervical afferent a x o n s from their cell bodies and only this procedure
Correspondence: T.H. Moran, Department of Psychiatry, Meyer 4-119, JohnsHopkins University School of Medicine; Baltimore, MD 21205, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
96 s h o u l d e l i m i n a t e C C K binding. A p r e l i m i n a r y r e p o r t of this w o r k has a p p e a r e d 2.
MATERIALS AND METHODS Male Sprague-Dawley rats served as the subjects for all experiments. Animals were maintained on a 12/12 h light-dark cycle and food was always available unless otherwise specified.
Supra- and infra-ganglionic vagotomies Animals were anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg, i.p.) and placed in a stereotaxic device in a supine position. A midline incision was made and the trachea exposed. The left cervical vagus was exposed and followed by blunt dissection along its course toward the posterior lacerated foramen. The nodose ganglion was identified under 16 power magnification. For supraganglionic vagotomies (n = 8, for central transport, n = 6, for peripheral transport) the ganglion was gently retracted from the foramen and its rostral extent identified. The tips of microscissors were placed along the foramen and the nerve cut above the ganglion. For infraganglionic vagotomies (n = 6), the nerve was severed approximately 0.5 cm distal to the nodose ganglion and retracted ventrally away from the foramen. The wound was closed with surgical clips and rats were returned to their home cages for 3-14 days depending on the particular experiment.
Subdiaphragmatic vagotomies Rats (n = 8) were food-deprived overnight and anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg, i.p.). Following a midline laparotomy, the stomach was placed under gentle retraction and the liver packed off with saline-soaked gauze, exposing the lower part of the thoracic esophagus. Using an operating microscope, the point of branching of the hepatic branch from the right subdiaphragmatic vagus was identified and the trunk proximal and distal to the branch was exposed and stripped of surrounding tissue. TWo ligations of 6-0 silk were placed around the vagal trunk approximately 0.5 cm apart. The areas between the ligations including the hepatic branch was excised and the wound was closed,
Rootlet transections Rats (n = 9) were food-deprived overnight, pretreated with atropine sulfate (0.1 mg/kg i,p.) and then anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg, i.p.). A deep level of anesthesia was maintained with supplemental doses of barbiturate (15 mg/kg) administered approximately every 60 min. Body temperature was monitored continuously and maintained at 37 + 1 °C with a heated surgical stage. A constant pressure respirator was available to relieve any respiratory distress. The surgical procedures were essentially the same as used in a previous publication z5 and will be recounted only briefly here. A detailed methodological paper is in preparation z3. Rats were placed in an atraumatic headholder, positioned supine, and a midline incision made to expose the trachea. After retracting the hyoid bone and the trachea, the base of the skull was exposed deep to the larynx by blunt dissection with the aid of an operating microscope. With the occipital bone cleared of muscle from the level of the bulla caudally to the hypoglossal canal, the jugular foramen was expanded medially with a dental burr and fine ronjeurs to expose the dura mater covering the medulla. Lancing the dura revealed the rootlets of the glossopharyngeal and vagus nerves, as well as the trunk of the accessory nerve. Intracranially, the vagus nerve separates into afferent rootlets, which reach the medulla dorsally, and efferent rootlets, which reach the medulla ventrally. Using fine forceps and iris scissors, the efferent rootlets including the accessory nerve were severed in 3 rats (1 left and 2 right); the afferent rootlets were cut in the 6 other animals (4 left, 2 right). The cranial opening was packed with gelfoam and the wound closed with surgical clips. The rats were then returned to their home cages for 3-10 days.
In order to confirm the rootlet cuts, 7 of the 9 animals were reanesthetized and the cervical vagus exposed on the operated side. Using a micromanipulator, the vagus was raised onto a small polyethylene platform and between 0.8 and 2.0 /~1 of 1.0% horseradish peroxidase conjugated to wheatgerm agglutinin (Sigma) was injected into the nerve via a stout glass pipette (100/~m tip diameter). After this brief (1 h) procedure, the rats were returned to their home cages for an additional 2-3 days, before being sacrificed for the receptor binding assay.
Vagal ligations For experiments addressing the peripheral transport of CCK receptors, 3 days following the supraganglionic and infraganglionic vagotomies, rats were reanesthetized and the cervical vagus exposed. The vagus was stripped of surrounding tissue and ligated with 5-0 silk at least 2 cm below the level of the nodose ganglion. The wound was closed with wound clips and the rats were returned to their home cages. Twenty-four hours later, animals were sacrificed by decapitation and a segment of the ligated nerve was removed. In order to maintain proper orientation, approximately 8 mm of nerve proximal and 4 mm of nerve distal to the ligature were taken. The ligature was removed and the nerve placed in the bottom of a paraffin boat on dry ice and covered with homogenized bovine brain paste. Embedded nerve segments were placed in plastic bags and stored at -70 °C. Ten/~m longitudinal sections were taken through the nerve segments at -15 °C, thaw-mounted onto gelatin-coated slides and dried in a desiccator under partial pressure at -4 °C. Slides were stored at -70 °C until binding procedures were performed.
Sacrifice and tissue sectioning For examining the effect of vagotomies on brainstem CCK receptors, the animals that did not receive HRP injections were sacrificed by decapitation from 5 to 14 days after the vagotomy procedures. The brain was rapidly removed, blocked behind the cortex and the brainstem including the cerebellum rapidly frozen in N-methyl-butane on dry ice. The rats that received vagal HRP injections were anesthetized as above and perfused through the heart with normal saline. Frozen brainstem blocks were placed in plastic bags and stored at -70 °C. Twenty ~m sections were taken through the extent of the NTS and mounted to gelatin-coated slides.
,~
Gelatinous
Subnucleus NTS Commissural NTS I ,',.,--" _,-~-:
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',
"
Posterior NTS
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.... J \ Fig. 1. Schematic representation of coronal caudal brainstem sections. The density of CCK binding was determined at each of the 3 anterior/posterior levels (gelatinous subnucleus, commissural and posterior NTS). Binding densities were compared in the medial and lateral cross hatched areas of the NTS and in the area postrema ipsilateral and contralateral to the vagal lesions. AP, area postrema; SolC, nucleus tractus solitarii, commissural; SolG, nucleus tractus solitarii, gelatinous; SolM, nucleus tractus solitarii, medial; SOIL, nucleus tractus solitarii, lateral; sol, solitary tract.
97 In the non-HRP animals sections were taken in pairs for receptor autoradiography. In HRP-treated animals, sections were taken in triplicate with one set used for HRP histochemistry.
Horseradish peroxidase histochemistry At least 2 slides from each brain were first fixed in 0.05%
glutaraldehyde for 1 h and the processed according to a protocol modified from Mesulam 7 and Gibson et al. 4 to reveal HRP reaction product 12. In this procedure, the sections were immersed serially in the solutions of tetramethyl benzidene and sodium nitroferricyanide for 5 or 8 sequences of 5 min duration. After the reaction process was completed, the sections were cleared in Histoclear, coverslipped
Fig. 2. Autoradiographic depiction of the effects of unilateral subdiaphragmatic (A, B and C) and r i o t supraganglionic vagotomy (D, E and F) at 3 anterior/posterior levels through the NTS and AP in representative animals.
98 and stored at 4 °C. In some preparations, the vagus and nodose ganglion on the operated side were removed and fixed 'en bloc' in 0.05% glutaraldehyde. Once fixed, this tissue was embedded in a gelatin-albuminmixture that was cured rapidly with a few drops of 10% glutaraldehyde. These blocks were then frozen, sectioned at 40 /~m, mounted on slides, and then processed following the same protocol used for the brain sections. Autoradiographic analysis of brainstem binding density was only carried out on rats whose rootlet lesions were substantiated by HRP transport.
AREA POSTREMA 10000
The slide-mounted tissue sections were incubated as previously described9-11 in buffer containing 50 pM [12sI]CCK-8(New England Nuclear). Alternate slides containing adjacent sections were incu-
Subdiaphragmatic Vagotomy ~l|nranannllnnlP. Vaootomu
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Fig. 4. Densities of [125I]CCK binding in the area postrema in response to unilateral subdiaphragmatic or supraganglionic vagotomies. Binding in the AP was not affected by either procedure.
bated in the presence of an excess (1 /~M) unlabelled CCK-8 to determine the extent of non-specific binding. Slides were washed with cold (4 °C) buffer and dried. Dried slides were placed in X-ray cassettes and oppossed to [3H]Ultrofilm (LKB) along with [141]standards (Amersham). Binding under the conditions used has been shown to be saturable, of high affinity, reversible, and characterized by a high ratio of specific to non-specific binding (85-90%). Autoradiographic images from the developed films were directly compared with the corresponding tissue sections, which had been stained with Cresyl violet, by viewing the superimposed autoradiograph and histologic section. This allowed accurate localization of CCK binding sites. Images were analyzed utilizing a computerized microdensitometry system (RAS-1000 Amersham). Specific binding was calculated as the difference in standard generated dpm's between adjacent sections incubated with and without 1 /~M unlabelled CCK-8.
Data analysis
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In the experiments examining the origin of NTS and AP CCK receptors, CCK binding density in the NTS was examined at 3 rostral caudal levels. At each level the NTS was divided into 4 quadrants and at the middle level, the AP was divided into 4 quadrants. These are demonstrated in Fig. 1. Data were analyzed by a mixed model ANOVA for the factors of vagotomy type and measurement side comparing regions ipsilateral and contralateral to the vagotomy procedures. A similar design was used for analyzing binding within AP. The AP was divided and the binding densities ipsilateral and contralaterai to the lesions were compared. Binding data for vagal segments were analyzed by ANOVA comparing the binding densities proximal and distal to the ligation for the two vagotomy procedures.
2000
RESULTS 1000
0
Lateral
Medial
CONTRALATERAL
Medial
Lateral
IPSILATERAL
Fig. 3. Densities of [1251]CCK binding in the lateral and medial aspects of the NTS at 3 anterior/posterior levels in response to unilateral subdiaphragmatic or supraganglionic vagotomies expressed as computer-generated DPMs/mg of tissue as a measure of binding density. Stars indicate significant depletions relative to contralateral levels. Supraganglionic but not subdiaphragmatic vagotomies affected binding densities.
Analysis of autoradiographs from rats receiving unilateral supraganglionic or unilateral subdiaphragmatic vagotomies revealed different patterns of [125I]CCK binding depending upon the type of vagotomy and the level of the NTS examined (Fig. 2). In the posterior NTS, supraganglionic vagotomy produced significant decreases in binding density ipsilateral to the lesion in both the medial and lateral aspects of the commissural NTS, expressed as significant interactions on the A N O V A
99 GELATINOUS SUBNUCLEUS 4OOO
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Fig. 5. Densities of [1251]CCK binding in the lateral and medial aspects of the NTS at 3 anterior/posteriorlevels followingunilateral efferent or afferent vagal rootlet transections. Stars indicate significant depletions relative to the contralateral levels. Afferent but not efferent rootlet transectionsproduce ipsilateral depletions in binding densities.
(lateral: El,14 = 35.308, P < 0.001; medial El,14 = 8.600, P < 0.01) (Fig. 3). At the level of the commissural NTS, supraganglionic vagotomy decreased binding density only in the lateral aspect ipsilateral to the lesion F1,14 = 6.21, P < 0.05. Supraganglionic vagotomy reduced binding density in the NTS at the level of the gelatinous subnucleus in both the ipsilateral medial aspect corresponding to the gelatinous and medial subnuclei (medial aspect: Fl,14 = 34.396, P < 0.001) and in the lateral aspect corresponding to the lateral subnuclei (lateral aspect: F1.14 = 5.614, P < 0.05). No changes in binding density were evident ipsilateral to the lesion in animals
with subdiaphragmatic vagotomies (Fig. 3). As well, no changes in binding density were found ipsilateral to the lesions in the AP in either group (Fig. 4). Unilateral efferent and afferent vagal rootlet transections differentially affected NTS CCK binding. HRP verification of the completeness of the lesions demonstrated that one animal with an efferent transection also had an afferent cut; in one afferent cut, the transection was incomplete in that there was significant label in the NTS. Data from these animals were not included in the analyses of binding density. Unilateral efferent transections had no effect on CCK binding at any level in either the medial or lateral aspects of the NTS (Fig. 5). In contrast, unilateral afferent vagal lesions produced significant decreases in CCK binding ipsilateral to the lesion at all levels of the NTS. ANOVA demonstrated a significant main effect of lesion in both the lateral and medial aspects of the NTS on the ipsilateral compared to contralateral side: lateral, F1,4 = 15.282, P < 0.02; medial, F1,4 = 7.888, P < 0.05. Planned t-analyses demonstrated significant depletions in binding density in the medial and lateral portions at all three levels of the NTS (Fig. 5). Neither efferent nor afferent rootlet transections produced ipsilateral depletions in the AP. A comparison of the unilateral afferent vagal input to the NTS as demonstrated by HRP transport in rats with unilateral efferent transections and the distribution of NTS CCK receptors in rats with unilateral afferent transections is demonstrated in Fig. 6. HRP verification of the lesion in rats receiving efferent transections demonstrated that unilateral afferent vagal inputs have both ipsilateral and contralateral projections in the NTS. The reaction product in NTS resulting from an HRP injection into the left cervical vagus overlaps substantially with the CCK receptor distribution remaining after a right vagal afferent rootlet transection (compare Fig. 6A, B and C with Fig. 6D, E and F, respectively). Thus, after a unilateral afferent rootlet transection, the high affinity CCK binding is confined to those areas of the NTS that are innervated by the intact, contralateral vagal afferent axons. Analysis of the effects of supra- and infraganglionic vagotomy on the transport of CCK binding sites in the cervical vagus revealed differential effects of the vagotomy procedures proximal but not distal to the vagal ligations, F1,10 = 5.273, P < 0.05. As demonstrated in Fig. 7, following supraganglionic vagotomy, vagal transport of CCK binding sites is intact as indicated by a significant increase in CCK binding proximal to the vagal ligature. This increase in binding density proximal to the ligature was eliminated by the infraganglionic vagotomy procedure.
100
Fig. 6. Comparison of unilateral vagal afferent input (A, B and C) as demonstrated by transganglionic HRP transport from the cervical vagus following a unilateral efferent rootlet transection and [125I]CCKbinding in a rat with a unilateral afferent rootlet transection (D, E and F) at 3 anterior/posterior levels in the NTS. The distribution of CCK binding sites following a unilateral afferent transection overlays the pattern of unilateral afferent vagal input.
DISCUSSION The results of these experiments demonstrate a number of phenomena. First, unilateral supraganglionic vagotomy produces a significant ipsilateral reduction in [125I]CCK binding in the NTS. This result is specific to severing the vagus above the level of the afferent cell bodies in the nodose ganglion as subdiaphragmatic
vagotomy does not alter CCK binding in the NTS. The specific vagal component underlying this transport is demonstrated by the finding that unilateral afferent rather than efferent rootlet transection results in unilateral reductions in CCK binding throughout the NTS. Together these findings demonstrate that a significant portion of CCK binding sites in the NTS are located presynaptically on vagal afferent terminals.
101 Vagal CCK Binding at Cervical L i g a t i o n Following Vagotomy 3000
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2000
•
Proximalto Ligation
•
Distal to Ltgatlon
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Fig. 7. Densitiesof [125I]CCKbindingproximal and distal to ligation of the cervical vagus in rats with either supraganglionic or infraganglioni¢ vagotomies. Significant levels of binding were present proximal to the ligationin rats with supraganglionic,but not infraganglionic, vagotomies. Furthermore, vagal transport of CCK binding sites toward peripheral axon terminals also occurs in afferent fibers because severing the vagus below the nodose ganglion, disconnecting axons from both efferent and afferent cell bodies interrupts this transport while severing the vagus above the nodose, disconnecting axons from only the efferent cell bodies does not. This finding, in combination with the central results demonstrates that the transport of CCK binding sites is bidirectional from afferent cell bodies in the nodose ganglion. In contrast, CCK binding in the area postrema was not affected by any of the vagotomy procedures. This result supports the view that binding sites in the AP are not of vagal origin. Although unilateral supraganglionic vagotomy caused a significant ipsilateral reduction in CCK binding in the NTS, this was a reduction rather than a unilateral elimination. This initial result complements that of Ladenheim, Speth and Ritter 7 with nodosectomy and the lack of a complete elimination may be interpreted in a number of ways. First, it is possible that a significant potion of CCK binding in the NTS is postsynaptic to vagal afferent axons and these sites are not affected by the vagotomy procedure. Alternatively, the extent of the elimination of NTS vagal afferent input produced by either lesion may not be complete. For example, the nodose ganglion does not have a sharp border. Afferent vagal cell bodies stream centrally away from the ganglion ] and either the supraganglionic vagotomy or the nodosectomy may leave a significant portion of afferent fibers connected to their cell bodies. Third, the remaining opposite intact unilateral input may project bilaterally in the NTS. The result of the afferent rootlet transection support the latter two interpretations. Following unilateral after-
ent rootlet transection the magnitude of ipsilateral depletion in CCK binding is somewhat greater than that achieved with supraganglionic vagotomy in that significant ipsilateral depletions are found at all levels of the NTS. This suggests that a greater number of afferent fibers may have been disconnected from their cell bodies with the rootlet transection procedure. In addition, as demonstrated by the HRP injections after efferent rootlet transections, the unilateral afferent vagal input into the NTS has a significant contralateral component. Furthermore, after a unilateral afferent transection, the distribution of NTS CCK receptors demonstrated a close correspondence to the pattern of HRP reaction product produced after labelling intact vagal afferent axons. Thus, although the question of whether all the CCK binding sites in the NTS are presynaptic and of vagal origin cannot be definitively answered from the present data, the patterns of depletion and afferent vagal innervation suggest that this may he the case. While supraganglionic vagotomy or afferent vagal transections affected [125I]CCK binding in the NTS, there were no unilateral reductions in binding density in the AP in response to these lesions. This is a similar result to that demonstrated by Ladenheim, Speth and Ritter 7 in response to unilateral nodosectomy. This result could be interpreted as indicating either that the vagal afferent input into the AP is completely bilateral or that these receptors are not on vagal afferent axons. The bilateral distribution interpretation is not supported by the data. This would imply that, after a unilateral transection the receptor density in the AP would be reduced by 50%. In fact, none of our experimental manipulations altered the density of CCK binding sites in the AP. Thus it appears that CCK binding sites in the AP are not of vagal origin. In as much as the AP is a circumventricular organ with a permeable blood brain barrier, the role of these receptors may be to respond to circulating CCK. The peripheral vagal transport of CCK receptors has been previously demonstrated in both the cervical vagus 19 and in all the subdiaphragmatic vagal branches 11. The movement of the receptors is by fast axonal transport in that it is blocked by colchicine ]9. Previous work, however, had not discerned whether transport occurred in the afferent or efferent limb of the vagus. In the present study, infraganglionic rather than supraganglionic vagotomy prevented the peripheral transport of CCK receptors in the cervical vagus as measured by an accumulation of CCK binding sites at a vagal ligation placed distal to vagal transection. Cell bodies for afferent vagal fibers are contained in the nodose ganglion while cell bodies for efferent vagal fibers are contained within the dorsal motor nucleus of the caudal hindbrain. Since both vagotomy procedures disconnect efferent vagal
102 fibers at the site of the ligation from their cell bodies, both would be expected to eliminate the peripheral transport of vagal C C K receptors if this were occurring in efferent vagal fibers. This was not the case. Only the infraganglionic vagotomy, which disconnected both afferent and efferent vagal fibers at the ligation point from their cell bodies, eliminated vagal C C K receptor transport. Thus this transport of C C K receptors occurs on vagal afferent fibers. The demonstration that vagal CCK receptors are contained in afferent rather than efferent fibers has important implications for the identification of potential sites of action for the feeding inhibitory action of CCK. Type A receptors are likely to mediate the feeding effects of CCK, but the particular receptor population at which this inhibitory action of C C K is mediated remains unclear. Candidate type A receptor populations have been identified in the circular muscle layer of the pyloric sphincter 17, as well as in the vagus and brain stem as discussed here. A role for the afferent vagus in the inhibition of food intake by CCK is evident from results demonstrating that total vagotomy or combined unilateral afferent rootlet transection and contralateral subdia-
phragmatic vagotomy eliminate C C K satiety aS. The present demonstration that it is the afferent vagal fibers which contain C C K receptors suggests that some aspect of CCK's feeding inhibitory action could be through the direct stimulation of these vagal C C K receptors. Circulating C C K may directly activate C C K receptors in the A P and on afferent vagal terminals 14. Circulating CCK is unlikely to have access to C C K receptors of vagal origin in the NTS. These receptors may play some role in modulating vagal afferent input in response to locally released CCK. Such a role is suggested in the work of Ewart and Wingate 3 demonstrating augmentation of NTS responses to gastric distention by iontophoretically applied CCK. Thus C C K receptors are present along the afferent vagus and at its central terminations but how actions of these various C C K receptor populations may interact in the inhibition of food intake by C C K remains to be determined.
REFERENCES
Cholecystokinin Antagonists, Alan R. Liss, New York, 1988, pp. 117-132. 10 Moran, T.H., Robinson, P.H., Goldrich, M.S. and McHugh, P.R., Two brain CCK receptors: implications for behavioral actions, Brain Research, 362 (1986) 175-179. 11 Moran, T.H., Smith, G.P., Hostetler, A.M. and McHugh, P.R., Transport of cholecystokinin (CCK) binding sites in subdiaphragmatic vagal branches, Brain Research, 415 (1987) 149-152. 12 Norgren, R. and Smith, G.P., Central distribution of subdiaphragmatic vagal branches in the rat, J. Comp. Neurol., 237 (1988) 207-223. 13 Norgren, R. and Smith, G.P., A method for selective section of vagal afferent or efferent axons in the rat, in preparation. 14 Raybould, H.E., Gayton, R.J. and Dockray, G.J., CNS effects of circulating CCK-8: involvement of brainstem neurons responding to gastric distention, Brain Research, 342 (1985) 187-190. 15 Smith, G.P., Jerome, C. and Norgren, R., Afferent axons of the abdominal vagus mediate the satiety effects of cholecystokinin in rats, Am. J. Physiol., 249 (1986) R638-R641. 16 Smith, G.P., Jerome, C. Cushin, B.J., Eterno, R. and Simansky, K.J., Abdominal vagotomy blocks the satiety effect of cholecystokinin in the rat, Science, 213 (1981) 1036-1037. 17 Smith, G.T., Moran, T.H., Coyle, J.T., Kuhar, M.J., O'Donahue, T.L. and McHugh, P.R., Anatomical localization of cholecystokinin receptors to the pyloric sphincter, Am. J. Physiol., 246 (1984) R127-130. 18 Zarbin, M.A., Innis, R.B., Wamsley, J.K., Snyder, S.H. and Kuhar, M.J., Autoradiographic localization of cholecystokinin receptors in rodent brain, J. Neurosci., 3 (1983) 877-906. 19 Zarbin, M.A., Wamsley, J.K., Innis, R.B. and Kuhar, M.J., Cholecystokinin receptors: presence and axonal flow in the rat vagus nerve, Life Sci., 29 (1981) 697-705.
1 Aitschuler, S.M., Xinman, B., Bieger, D., Hopkins, D.A. and Miselis, R.M., Viscerotopic representation of the upper alimentary tract in the rat: sensory ganglia and nuclei of the solitary and spinal trigeminal tracts, J. Comp. Neurol., 283 (1989) 248-268. 2 Crosby, R.J., Norgren, R., Moran, T.H. and McHugh, P.R., Central and peripheral transport of CCK-A receptors on vagal afferent fibers, FASEB J., 3 (1989) A998. 3 Ewart, W.R. and Wingate, D.L., Cholecystokinin octapeptide and gastric mechanoreceptor activity in rat brain, Am. J. Physiol., 244 (1983) G613-G617. 4 Gibson, A., Hansma, D., Houk, J. and Robinson, E, A sensitive low artifact TMB procedure for the demonstration of WGA-HRP in the CNS, Brain Research, 298 (1984) 235-241. 5 Hill, D.R., Campbell, N.J., Shaw, T.M. and Woodruff, G.N., Autoradiographic localization and biochemical characterization of peripheral type CCK receptors in the rat CNS using highly specific nonpeptide CCK antagonists, J. Neurosci., 7 (1987) 2967-2976. 6 Kalia, M. and Sullivan, J.M., Brain stem projections of sensory and motor components of the vagus nerve, J. Comp. Neurol., 211 (1982) 248-264. 7 Ladenheim, E.E., Speth, R.C. and Ritter, R.C., Reduction of CCK-8 binding in the nucleus of the solitary tract in unilaterally nodosectomized rats, Brain Research, 474 (1988) 125-129. 8 Mesulam, M.M., Tetramethylbenzidene for horseradish peroxidase neurochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing afferent and efferents, J. Histochem. Cytochem., 26 (1978) 106-117. 9 Moran, T.H. and McHugh, P.R., Anatomical and pharmacological differentiation of pyloric, vagal and brainstem cholecystokinin receptors. In R.Y. Wang and R. Schoenfeld (Eds.),
Acknowledgements. This work was supported by NIH Grants DK 19302 and DC 00240 and generous gifts from Mrs. Samuel Hecht and the Lorraine and Leonard Levin Fund for Research in Biological Psychiatry. R.N. is a recipient of an NIMH Research Scientist Award, Level If (MH 00653).
