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Cholecystokinin-octapeptide like immunoreactivity in the area postrema of the rat and cat
Regulatory Peptides, 13 (1985) 31-40
31
Elsevier RPT 00435
Cholecystokinin-octapeptide like immunoreactivity in the area postrema of the rat and cat Bruce W. Newton* and Bruce E. Maley Department of Anatomy, University of Kentucky Medical Center, 800 Rose Street, Lexington, Kentucky 40536, U.S.A. (Received 2 July 1985; revised manuscript received 26 August 1985; accepted for publication 24 September 1985)
Summary The distribution of cholecystokinin-8 (CCK-8)-like immunoreactivity in the area postrema of the rat and cat was visualized using the peroxidase, antiperoxidase technique. In the rat the greatest amount of immunostaining occurred in peripheral regions of the area postrema at intermediate and rostral levels. Caudally, scattered immunoreactivity predominated. After colchicine treatment, numerous immunoreactive somata were observed throughout the area postrema. The cat area postrema had a different and more complex pattern of immunostaining than the rat. Moderate to dense accumulations of immunostaining occurred in the ventromedial region of the area postrema bordering the solitary tract and dorsal vagal nuclei. The central region of the area postrema possessed scattered amounts of immunoreactivity at rostral levels. Following colchicine treatment, no visible CCK-8-1ike immunoreactive cell bodies were observed in the cat area postrema. Results of the present investigation provide morphological evidence for the role of CCK-8 in cardiovascular regulation and satiety. The difference in the distribution of CCK-8 in the rat and cat suggest a possible role in the emetic reflex. circumventricular organ; neuropeptide; satiety; putative neurotransmitter; emesis
* To whom correspondence should be addressed. Present address: Department of Neurology, Monroe Community Hospital, 435 E. Henrietta Road, Rochester, NY 14603, U.S.A. Abbreviations: AP, area postrema; CC, central canal; Gr, gracile nucleus; IV, fourth ventricle; NTS, nucleus tractus solitarius; V, vestibular nucleus; DMN, dorsal motor nucleus of the vagus. 0167-0115/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
32 Introduction
The area postrema (AP), a circumventricular organ located on the dorsal surface of the brainstem at the level of the obex, is a vascular rich organ lying outside the blood-brain barrier. Consequently intrinsic AP elements and afferents projecting to the AP are exposed to both cerebrospinal fluid and plasma components [1]. In species possessing an emetic reflex, e.g. the cat, the AP is a 'chemoreceptor trigger zone' for the emetic center. In species without the emetic reflex, e.g. the rat, the AP has been associated with the acquisition of taste aversion learning [2,3] and feeding behavior [4]. In both emetic and non-emetic species the AP, along with the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMN), is involved with cardiovascular control [5,6]. The AP of both rat and cat has reciprocal connections with NTS and DMN [7-9], as well as receiving primary vagal afferent projections [10-121. Cholecystokinin-octapeptide (CCK-8) has a number of proposed functions within the central nervous system. The immunohistochemical localization of CCK-8 in the NTS and D M N strongly suggests that it is involved with cardiovascular and respiratory functions [13-15]. CCK-8 has also been shown to be a satiety factor [16,17] whose sites of action are not presently known. Additionally, investigations have established its role as a putative neurotransmitter of certain vagus nerve fibers [18,19]. It has been demonstrated that the AP contains the highest number of CCK-8 binding sites in the brainstem of the rat [20]. Although the immunohistochemical distribution of CCK-8 within the central nervous system of the rat has been described by many investigators [14,21-23], to date there has not been a detailed immunohistochemical description of this putative neurotransmitter within the AP of the rat. Furthermore, no investigations describe this peptide within the cat AP. This study is the first to describe in detail the distribution of CCK-8-1ike immunoreactive (CCK-8-LI) fibers and cell bodies within the AP of the rat and cat. These findings provide morphological evidence for the possible action of CCK-8 within the AP of these two species.
Materials and Methods
The present investigation utilized 12 Sprague-Dawley rats (6 untreated; 6 colchicine-treated) and 6 random bred cats (4 untreated; and 2 colchicine-treated). Colchicine treatment, following deep anesthesia with sodium pentobarbitol (50 mg/kg per rat; 38 mg/kg per cat), consisted of an intracisternal injection of colchicine (65 #g/rat; 250/~g/cat) in physiological saline and then followed by a 48 h survival period. Subsequently, animals were deeply anesthesized as before (cats were also intubated and artificially ventilated), a thoracotomy was performed, injected intracardially with heparin (10 units/rat; 100 units/cat), and perfused with physiological saline followed by 4% paraformaldehyde in Sorenson's phosphate buffer, pH 7.2 (650 ml/rat; 2.0 1/cat) at room temperature. The brains were left in situ for 1 h, then removed and placed in fresh fixative for an additional 90 min. Following this brief post-fixation, the brains were transferred to 20% phosphate buffered sucrose (0.1 M, pH 7.2) at
33 4"C. CCK-8-LI in the AP was visualized using the peroxidase-antiperoxidase (PAP) technique [24] as previously described [25]. The CCK-8 antiserum was obtained from the Immunonuclear Corporation, Stillwater, Minnesota. To test for the specificity of the CCK-8 antiserum, adjacent sections of the brainstem containing the AP were incubated with antiserum 'absorbed' with the CCK-8 peptide (10/~g/ml) 48 h prior to its use. The control sections exposed to this 'absorbed' antiserum were processed in parallel with the remainder of the sections and in all cases the antigen prevented immunostaining in the control sections. To evaluate the CCK-8-LI, brainstem sections containing the AP were projected onto paper and the outlines of representative caudal to rostral levels of the AP and neighboring nuclei were traced. The relative densities of CCK-8-LI were subsequently evaluated and rated on a scale of dense, moderate, scattered or no visible immunoreactivity by several investigators and mapped onto the AP line drawings. The distribution of CCK-8-LI fibers and cell bodies is symmetrical in each species and the line drawings are representative of the immunoreactivity observed in all experimental animals.
Results Rat
The distribution of CCK-8-LI fibers and cell bodies in the AP of the rat is summarized in Fig. 1A-C (caudal to rostral). In general, the greatest accumulations of CCK-8-LI fibers occurred in the AP at intermediate levels (Fig. 1B) and decreased in both caudal and rostral directions (Fig. 1A and C). At the intermediate level the majority of CCK-8-LI fibers occupied the dorsal, ventrolateral and ventral borders of the AP, while the central region had only scattered immunoreactive fibers (Figs. 1B and 2A and B). In caudal to intermediate levels the ventral and ventrolateral borders possessed scattered immunoreactivity, while the dorsal surface completely lacked CCK-8-LI staining (Fig. IA). Interestingly, the central region, which had very little CCK-8-LI fibers at intermediate levels, contained moderate amounts of CCK8-LI fibers at caudal levels. In contrast, the central region of the AP at a rostral level lacked CCK-8-LI staining (Fig. 1C). The distribution of CCK-8-LI fibers along the border of the rostral AP varied from dense accumulations at the ventrolateral area to moderate and scattered immunoreactivity in the dorsal and ventral border. Although some CCK-8-LI cell bodies were visible in untreated rats, colchicinetreatment revealed a greater number of more intensely immunostained cell bodies. CCK-8-LI cell body numbers were greatest at intermediate AP levels, and decreased both caudal- and rostralward (Fig. 1A-C). Most CCK-8-LI cell bodies tended to be located near the ventrolateral and ventral regions of the AP (Fig. 2C), although some immunostained neurons were present in central and dorsal areas. The CCK-8-LI cell bodies appeared most often as fusiform cells with a diameter of approximately 15 #m (Fig. 2D). Immunostained processes originating from the soma were of variable length, and occasionally bifurcated (Fig. 2D). At most levels AP CCK-8-LI processes often crossed the AP-NTS boundary (Fig. 2 D).
34
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Fig. 1. Line drawings (A-C, caudal to rostral) of the dorsal portion of the rat caudal brainstem at the level of the midline area postrema. The densities of immunoreactive fibers within the AP following incubation with CCK-8 antiserum are indicated on the right half of the line drawing. Densities were classified as dense (D), moderate (M), scattered (S), or none (N). CCK-8-LI cell bodies in the AP are shown on the left half of the line drawing with each point corresponding to one cell body. Line drawings (D-F, caudal to rostral) of the right side of the dorsal portion of the cat caudal brainstem. The densities of immunoreactive fibers within the AP following incubation with CCK-8 antiserum are indicated and classified as dense (D), moderate (M), scattered (S), or none (N). CCK-8-LI cell bodies were not present in the AP of untreated or colchicine-treated cats.
Fig. 2. (A) Photomicrograph of CCK-8-LI immunoreactivity in the rat AP at an intermediate level. Note the immunoreactivity along the ventrolateral and ventral boundaries (arrows) and the lack of immunostaining in central regions (asterisk). x 66. (B) Higher magnification of the lateral border in (A). CCK-8LI is contained within fibers (arrows) and a few cell bodies (arrowheads). x 198. (C) Photomicrograph of CCK-8-LI cell bodies in the lateral region of the AP. Numerous cell bodies immunostained for CCK-8 are also visible in the NTS. Dashed line separates NTS from AP. × 236. (D) Photomicrograph of CCK8-LI cells along the ventral boundary of the AP. The cells have dendrites which extend into the NTS. Dashed line separates AP from the NTS. × 540.
37 Cat The distribution o f CCK-8-LI fibers within the AP of the cat is summarized in Fig. 1 (D-F, caudal to rostral). The AP of the cat contained fewer CCK-8-LI fibers and in addition, had a distribution different from that of the rat. Unlike the rat, CCK8-LI cell bodies were not present in either untreated or colchicine-treated cats. In general, the numbers of CCK-8-LI fibers decreased from caudal to rostral levels (Fig. 1D-F). The greatest accumulation of CCK-8-LI fibers occurred in the ventromedial region o f the caudal and intermediate AP (Figs. 1D-E; 3A and B). At more rostral level (Fig. 1F) this dense pattern was replaced with scattered to moderate accumulations of CCK-8-LI fibers. The remainder of the caudal AP consisted of a patchwork of scattered to dense CCK-8-LI fibers. At a more rostral level the majority o f the AP was occupied by scattered amounts of CCK-8-LI fibers although moderate accumulation of CCK-8-LI fibers could be found near the ventral and lateral AP borders. At intermediate levels a 'band' of increased immunoreactivity was observed that originated in the ventromedial AP region near the borders of the NTS and D M N (Fig. 3C). This 'band' o f CCK-8-LI fibers swept dorsally and laterally through the medial-half of the AP before diminishing.
Discussion This study provides a detailed immunohistochemical description of CCK-8-LI fibers and cell bodies within the rat AP, and the first visualization of CCK-8-LI fibers within the AP of the cat. CCK-8-LI fibers and cell bodies were found to be differentially distributed in the AP of both species. In the rat AP, CCK-8-LI fibers were most abundant at intermediate levels, whereas in the cat AP, the greatest accumulation of CCK-8-LI fibers were present caudally. In both species the majority of CCK-8-LI fibers were located along the borders of the AP. Large numbers of CCK-8-LI cell bodies were present in the AP o f colchicine-treated rats, but were lacking in the AP o f both untreated and colchicine-treated cats. It is possible that the amount of colchicine administered to the cats was not sufficient to reveal CCK-8-LI cell bodies. However, this is unlikely since additional cats treated with colchicine at levels (200 #g/kg body weight) comparable to that given to rats did not have CCK-8-LI cell bodies in the AP (unpublished observations). The results presented here support and expand upon previous studies that have reported CCK-8-LI fibers within the AP of the rat using PAP and immunohistofluorescent techniques [14,15,22]. In the rat AP, CCK-8-LI fibers probably originate from both intrinsic and extrinsic sources. CCK-8-LI AP neurons provide a portion of these fibers, but a number of them may originate from fibers crossing the AP-NTS
4 Fig. 3. (A) Photomicrographof CCK-8-L1fibersin the AP of the cat. The majority ofimmunostaining is located along the ventromedial border (asterisk). x 60. (B) Photomicrograph of the dense band of CCK-8-LI fibersin the ventromedialregion of the AP (arrow). The CCK-8-LI fibersdecreasein numbers in more dorsal and lateral regions, x 145. (C) Photomicrograph of CCK-8-LI fibers in the dorsal region of the AP. A band of immunoreactivity(arrow) sweeps through this area. x 155.
38 border from CCK-8-LI somata lying within the NTS and DMN [14,15,22]. Another possible source for AP CCK-8-LI fibers are from CCK-8-LI afferents from the vagus nerve [18,19]. Because the cat AP lacks CCK-8-LI somata, CCK-8-LI fibers are necessarily of extrinsic origin, probably arising from CCK-8-LI cell bodies that lie within the NTS (unpublished observations), or from CCK-8-LI vagal afferents [18,19]. Recently, new information has been gathered concerning the role of the AP in cardiovascular function [5,6], and CCK-8 may play a part in this role. CCK-8 has been demonstrated to be involved with the cardiovascular and respiratory functions of the NTS [13,15,26]. Because the AP has extensive reciprocal connections with the NTS and DMN [8,9] a portion of the CCK-8-LI fibers observed in these two nuclei may be of AP origin. If this is the case, then AP CCK-8-LI fibers projecting to the NTS and DMN may be involved in the regulation of cardiovascular control. However, partial cardiovascular control may be intrinsic to the AP. CCK-8-LI fibers and cell bodies are found in greatest numbers around the periphery of the AP; a region that is rich in primary vagal afferents [12,17,27]. Additionally, other neuropeptides involved with cardiovascular regulation, such as substance P and enkephalins, are also found within the AP. These AP substance P and enkephalin immunoreactive fiber patterns overlap and are also most numerous around the periphery of the AP [28]. Closely overlapping distributions of substance P and enkephalin immunoreactivities have also been observed in cardiovascular divisions of the NTS [29,30] which receive large numbers of vagal afferent projections [10-12]. Therefore, similar AP neurotransmitter distribution patterns, associated with terminal fields from vagal afferents, suggest that peripheral AP regions may be a site of autonomic control. Although it has been shown by several investigators that CCK-8 acts as a satiety agent [16,17] the locus of this action has not been elucidated. Researchers [31] have demonstrated that gastric vagotomy attenuates the reduced feeding response induced by exogenous CCK-8, suggesting that CCK-8 acts via a vagal influence. However, intracerebroventricular injections of CCK-8 also reduce feeding [16], suggesting a central site of action. In a recent study van der Kooy [32] found that AP lesions in rats attenuate the satiety effects of exogenous CCK-8. Therefore, the AP may be a site where CCK-8 acts to reduce food intake via a CCK-8 induced malaise. This hypothesis takes into account both of the previously observed vagal and central satiety effects of CCK-8. Accordingly, ablation of the AP will result in the loss of CCK-8 vagal afferents [12,18,19,27], eliminating a peripheral CCK-8 effect, as well as destroying AP CCK-8 receptors [20], eliminating a central CCK-8 effect. The immunohistochemical results presented in our study provide a morphological basis for this hypothesis. Our results demonstrate, at least in the rat, that a portion of the CCK-8-LI in the AP is of an intrinsic nature rather than being solely derived from CCK-8-LI vagal afferents. Although the influences of the vagus in CCK-8 dependent satiety can not be ignored, the large number of CCK-8-LI somata and fibers present in the AP indicate that the AP is a likely site where CCK-8 can induce satiety. To what degree these intrinsic CCK-8-LI AP neurons contribute to satiety is presently not known, but should be considered in future satiety experiments that utilize rats as an experimental model.
39
The differential distribution of CCK-8-LI between emetic and non-emetic species may be related to the ability of a species to demonstrate this reflex behavior. Differences in the distribution of other putative neurotransmitters between emetic and non-emetic species, such as FMRFamide, serotonin, enkephalin, and substance P have also been noted [25,28]. However, more emetic and non-emetic species will have to be examined before a trend can be established that correlates putative neurotransmitter distribution with the emetic reflex.
References 1 Borison, H.L., History and status of the area postrema, Fed. Proc., 43 (1984) 2937-2940. 20ssenkopp, K.-P., Taste aversions conditioned with gamma radiation: attenuation by area postrema lesions in rats, Behav. Brain Res,, 7 (1983) 297-305. 3 Ritter, S., McGlone, J.L. and Kelley, K.W., Absence of lithium-induced taste aversion after area postrema lesion, Brain Res., 201 (1980) 501-506. 4 Contreras, R.J., Kosten, T. and Bird, E., Area postrema: part of the autonomic circuitry of caloric homeostasis, Fed. Proc., 43 (1984) 2966-2968. 5 Barnes, K.L., Ferrario, C.M., Chernicky, C.L. and Brosnihan, K.B., Participation of the area postrema in cardiovascular control in the dog, Fed. Proc., 43 (1984) 2959-2962. 6 Szilagyi, J.E. and Ferrario, C.M., Central opiate system modulation of the area postrema pressor pathway, Hypertension, 3 (1981) 313-317. 7 Leslie, R.A. and Gwyn, D.G., Neuronal connections of the area postrema, Fed. Proc., 43 (1984) 2941-2943. 8 Van der Kooy, D. and Koda, L.Y., Organization of the projections of a circumventricular organ: the area postrema in the rat, J. Comp. Neurol., 219 (1983) 328-338. 9 Vigier, D. and Rouviere, A., Afferent and efferent connections of the area postrema demonstrated by the horseradish peroxidase method, Arch. Ital. Biol., 117 (1979) 325-339. 10 Kalia, M. and Mesulam, M.-M., Brain stem projections of sensory and motor components of the vagus complex in the cat. I. The cervical vagus and nodose ganglion, J. Comp. Neurol., 193 (1980) 435-465. 11 Kalia, M. and Mesulam, M.-M., Brain stem projections of sensory and motor components of the vagus complex in the cat. II. Laryngeal, tracheobronchial, pulmonary, cardiac, and gastrointestinal branches, J. Comp. Neurol., 193 (1980) 467-508. 12 Leslie, R.A., Gwyn, D.G. and Hopkins, D.A., The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat, Brain Res. Bull., 8 (1982) 37-43. 13 Morin, M.P., DeMarchi, P., Champaguat, J., Vanderhaeghen, J.J., Rossier, J. and Denavit-Saubie, M., Inhibitory effect of cholecystokinin octapeptide on neurons in the nucleus tractus solitarius, Brain Res., 265 (1983) 333-338. 14 Palkovits, M., Kiss, J.S., Beinfeld, M.C. and Williams, T.H., Cholecystokinin in the nucleus of the solitary tract of the rat: evidence for its vagal origin, Brain Res., 252 (1982) 386-390. 15 Takagi, H., Kubota, Y., Mori, S., Tateishi, K., Hamaoka, T. and Tohyama, M., Fine structural studies of cholecystokinin-8-1ike immunoreactive neurons and axon terminals in the nucleus of tractus solitarius of the rat, J. Comp. Neurol., 227 (1984) 369-379. 16 Della-Fera, M.A. and Baile, C.A., Cholecystokinin octapeptide: continuous picomole injections into the cerebral ventricles of sheep suppress feeding, Science, 206 (1979) 471-473. 17 Gibbs, J., Young, R.C. and Smith, G.P., Cholecystokinin elicits satiety in rats with open gastric fistulas, Nature, 245 (1973) 323-325. 18 Rehfeld, J.F., Cholecystokinin, Trends Neurosci., 3 (1980) 65-67. 19 Rehfeld, J.F., Gastrin and cholecystokinin in the vagus, J. Autonom. Nerv. Syst., 9 (1983) 113-118. 20 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.
40 21 Innis, R.B., Correa, F.M.A., Uhl, G.R., Schneider, B. and Snyder, S.H., Cholecystokinin octapeptide-like immunoreactivity: histochemical localization in rat brain, Proc. Natl. Acad. Sci. USA, 76 (1979) 521-525. 22 Kiyama, H., Shiosaka, S., Kubota, Y., Cho, H.J., Takagi, H., Tateishi, K., Hasimura, E., Hamaoka, T. and Tohyama, M., Ontogeny of cholecystokinin-8 containing neuron system of the rat: an immunohistochemical analysis. 1I. Lower brain stem, Neuroscience, 10 (1983) 1341-1359. 23 Kubota, Y., Inagaki, S., Shiosaka, S., Cho, H.J., Tateishi, K., Hasimura, E., Hamaoka, T. and Tohyama, M., The distribution of cholecystokinin octapeptide-like structures in the lower brain stem of the rat: an immunohistochemical analysis, Neuroscience, 9 (1983) 587-604. 24 Sternberger, L.A., Immunocytochemistry, Prentice-Hall, Englewood Cliffs, NJ, 1974. 25 Newton, B.W., Maley, B., Sasek, C. and Traurig, H., Distribution of FMRF-NH2-1ike immunoreactivity in rat and cat area postrema, Brain Res. Bull., 12 (1984) 391-399. 26 Pagani, F.D., Taveira de Silva, A.M., Hamosh, P., Garvey, TQ., III and Gillis, R.A., Respiratory and cardiovascular effects of intraventricular cholecystokinin, Eur. J. Pharmacol., 78 (1982) 129-132. 27 Kalia, M. and Sullivan, J.M., Brainstem projections of sensory and motor components of the vagus nerve in the rat, J. Comp. Neurol., 211 (1982) 248-264. 28 Newton, B.W., Maley, B. and Traurig, H., The distribution of substance P, enkephalin, and serotonin immunoreaetivities in the area postrema of the rat and cat, J. Comp. Neurol., 234 (1985) 87-104. 29 Kalia, M., Fuxe, K., Hrkfelt, T., Johansson, O., Lang, R., Ganten, D., Cueilo, C. and Terenius, L., Distribution of neuropeptide immunoreactive nerve terminals within the subnuclei of the nucleus of the tractus solitarius of the rat, J. Comp. Neurol., 222 (1984) 409--444. 30 Maley, B. and Elde, R., Immunohistochemical localization of putative neurotransmitters within the feline nucleus tractus solitarii, Neuroscience, 7 (1982) 2469-2490. 31 Smith, G.P., Herome, C., Cushion, B.J., Eterno, R. and Simansky, K.J., Abdominal vagotomy blocks the satiety effect of cholecystokinin in the rat, Science, 213 (1981) 1036-1037. 32 Van der Kooy, D., Area postrema: site where cholecystokinin acts to decrease food intake, Brain Res., 295 (1984) 345-347.
31
Elsevier RPT 00435
Cholecystokinin-octapeptide like immunoreactivity in the area postrema of the rat and cat Bruce W. Newton* and Bruce E. Maley Department of Anatomy, University of Kentucky Medical Center, 800 Rose Street, Lexington, Kentucky 40536, U.S.A. (Received 2 July 1985; revised manuscript received 26 August 1985; accepted for publication 24 September 1985)
Summary The distribution of cholecystokinin-8 (CCK-8)-like immunoreactivity in the area postrema of the rat and cat was visualized using the peroxidase, antiperoxidase technique. In the rat the greatest amount of immunostaining occurred in peripheral regions of the area postrema at intermediate and rostral levels. Caudally, scattered immunoreactivity predominated. After colchicine treatment, numerous immunoreactive somata were observed throughout the area postrema. The cat area postrema had a different and more complex pattern of immunostaining than the rat. Moderate to dense accumulations of immunostaining occurred in the ventromedial region of the area postrema bordering the solitary tract and dorsal vagal nuclei. The central region of the area postrema possessed scattered amounts of immunoreactivity at rostral levels. Following colchicine treatment, no visible CCK-8-1ike immunoreactive cell bodies were observed in the cat area postrema. Results of the present investigation provide morphological evidence for the role of CCK-8 in cardiovascular regulation and satiety. The difference in the distribution of CCK-8 in the rat and cat suggest a possible role in the emetic reflex. circumventricular organ; neuropeptide; satiety; putative neurotransmitter; emesis
* To whom correspondence should be addressed. Present address: Department of Neurology, Monroe Community Hospital, 435 E. Henrietta Road, Rochester, NY 14603, U.S.A. Abbreviations: AP, area postrema; CC, central canal; Gr, gracile nucleus; IV, fourth ventricle; NTS, nucleus tractus solitarius; V, vestibular nucleus; DMN, dorsal motor nucleus of the vagus. 0167-0115/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
32 Introduction
The area postrema (AP), a circumventricular organ located on the dorsal surface of the brainstem at the level of the obex, is a vascular rich organ lying outside the blood-brain barrier. Consequently intrinsic AP elements and afferents projecting to the AP are exposed to both cerebrospinal fluid and plasma components [1]. In species possessing an emetic reflex, e.g. the cat, the AP is a 'chemoreceptor trigger zone' for the emetic center. In species without the emetic reflex, e.g. the rat, the AP has been associated with the acquisition of taste aversion learning [2,3] and feeding behavior [4]. In both emetic and non-emetic species the AP, along with the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMN), is involved with cardiovascular control [5,6]. The AP of both rat and cat has reciprocal connections with NTS and DMN [7-9], as well as receiving primary vagal afferent projections [10-121. Cholecystokinin-octapeptide (CCK-8) has a number of proposed functions within the central nervous system. The immunohistochemical localization of CCK-8 in the NTS and D M N strongly suggests that it is involved with cardiovascular and respiratory functions [13-15]. CCK-8 has also been shown to be a satiety factor [16,17] whose sites of action are not presently known. Additionally, investigations have established its role as a putative neurotransmitter of certain vagus nerve fibers [18,19]. It has been demonstrated that the AP contains the highest number of CCK-8 binding sites in the brainstem of the rat [20]. Although the immunohistochemical distribution of CCK-8 within the central nervous system of the rat has been described by many investigators [14,21-23], to date there has not been a detailed immunohistochemical description of this putative neurotransmitter within the AP of the rat. Furthermore, no investigations describe this peptide within the cat AP. This study is the first to describe in detail the distribution of CCK-8-1ike immunoreactive (CCK-8-LI) fibers and cell bodies within the AP of the rat and cat. These findings provide morphological evidence for the possible action of CCK-8 within the AP of these two species.
Materials and Methods
The present investigation utilized 12 Sprague-Dawley rats (6 untreated; 6 colchicine-treated) and 6 random bred cats (4 untreated; and 2 colchicine-treated). Colchicine treatment, following deep anesthesia with sodium pentobarbitol (50 mg/kg per rat; 38 mg/kg per cat), consisted of an intracisternal injection of colchicine (65 #g/rat; 250/~g/cat) in physiological saline and then followed by a 48 h survival period. Subsequently, animals were deeply anesthesized as before (cats were also intubated and artificially ventilated), a thoracotomy was performed, injected intracardially with heparin (10 units/rat; 100 units/cat), and perfused with physiological saline followed by 4% paraformaldehyde in Sorenson's phosphate buffer, pH 7.2 (650 ml/rat; 2.0 1/cat) at room temperature. The brains were left in situ for 1 h, then removed and placed in fresh fixative for an additional 90 min. Following this brief post-fixation, the brains were transferred to 20% phosphate buffered sucrose (0.1 M, pH 7.2) at
33 4"C. CCK-8-LI in the AP was visualized using the peroxidase-antiperoxidase (PAP) technique [24] as previously described [25]. The CCK-8 antiserum was obtained from the Immunonuclear Corporation, Stillwater, Minnesota. To test for the specificity of the CCK-8 antiserum, adjacent sections of the brainstem containing the AP were incubated with antiserum 'absorbed' with the CCK-8 peptide (10/~g/ml) 48 h prior to its use. The control sections exposed to this 'absorbed' antiserum were processed in parallel with the remainder of the sections and in all cases the antigen prevented immunostaining in the control sections. To evaluate the CCK-8-LI, brainstem sections containing the AP were projected onto paper and the outlines of representative caudal to rostral levels of the AP and neighboring nuclei were traced. The relative densities of CCK-8-LI were subsequently evaluated and rated on a scale of dense, moderate, scattered or no visible immunoreactivity by several investigators and mapped onto the AP line drawings. The distribution of CCK-8-LI fibers and cell bodies is symmetrical in each species and the line drawings are representative of the immunoreactivity observed in all experimental animals.
Results Rat
The distribution of CCK-8-LI fibers and cell bodies in the AP of the rat is summarized in Fig. 1A-C (caudal to rostral). In general, the greatest accumulations of CCK-8-LI fibers occurred in the AP at intermediate levels (Fig. 1B) and decreased in both caudal and rostral directions (Fig. 1A and C). At the intermediate level the majority of CCK-8-LI fibers occupied the dorsal, ventrolateral and ventral borders of the AP, while the central region had only scattered immunoreactive fibers (Figs. 1B and 2A and B). In caudal to intermediate levels the ventral and ventrolateral borders possessed scattered immunoreactivity, while the dorsal surface completely lacked CCK-8-LI staining (Fig. IA). Interestingly, the central region, which had very little CCK-8-LI fibers at intermediate levels, contained moderate amounts of CCK8-LI fibers at caudal levels. In contrast, the central region of the AP at a rostral level lacked CCK-8-LI staining (Fig. 1C). The distribution of CCK-8-LI fibers along the border of the rostral AP varied from dense accumulations at the ventrolateral area to moderate and scattered immunoreactivity in the dorsal and ventral border. Although some CCK-8-LI cell bodies were visible in untreated rats, colchicinetreatment revealed a greater number of more intensely immunostained cell bodies. CCK-8-LI cell body numbers were greatest at intermediate AP levels, and decreased both caudal- and rostralward (Fig. 1A-C). Most CCK-8-LI cell bodies tended to be located near the ventrolateral and ventral regions of the AP (Fig. 2C), although some immunostained neurons were present in central and dorsal areas. The CCK-8-LI cell bodies appeared most often as fusiform cells with a diameter of approximately 15 #m (Fig. 2D). Immunostained processes originating from the soma were of variable length, and occasionally bifurcated (Fig. 2D). At most levels AP CCK-8-LI processes often crossed the AP-NTS boundary (Fig. 2 D).
34
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F RAT
CAT
Fig. 1. Line drawings (A-C, caudal to rostral) of the dorsal portion of the rat caudal brainstem at the level of the midline area postrema. The densities of immunoreactive fibers within the AP following incubation with CCK-8 antiserum are indicated on the right half of the line drawing. Densities were classified as dense (D), moderate (M), scattered (S), or none (N). CCK-8-LI cell bodies in the AP are shown on the left half of the line drawing with each point corresponding to one cell body. Line drawings (D-F, caudal to rostral) of the right side of the dorsal portion of the cat caudal brainstem. The densities of immunoreactive fibers within the AP following incubation with CCK-8 antiserum are indicated and classified as dense (D), moderate (M), scattered (S), or none (N). CCK-8-LI cell bodies were not present in the AP of untreated or colchicine-treated cats.
Fig. 2. (A) Photomicrograph of CCK-8-LI immunoreactivity in the rat AP at an intermediate level. Note the immunoreactivity along the ventrolateral and ventral boundaries (arrows) and the lack of immunostaining in central regions (asterisk). x 66. (B) Higher magnification of the lateral border in (A). CCK-8LI is contained within fibers (arrows) and a few cell bodies (arrowheads). x 198. (C) Photomicrograph of CCK-8-LI cell bodies in the lateral region of the AP. Numerous cell bodies immunostained for CCK-8 are also visible in the NTS. Dashed line separates NTS from AP. × 236. (D) Photomicrograph of CCK8-LI cells along the ventral boundary of the AP. The cells have dendrites which extend into the NTS. Dashed line separates AP from the NTS. × 540.
37 Cat The distribution o f CCK-8-LI fibers within the AP of the cat is summarized in Fig. 1 (D-F, caudal to rostral). The AP of the cat contained fewer CCK-8-LI fibers and in addition, had a distribution different from that of the rat. Unlike the rat, CCK8-LI cell bodies were not present in either untreated or colchicine-treated cats. In general, the numbers of CCK-8-LI fibers decreased from caudal to rostral levels (Fig. 1D-F). The greatest accumulation of CCK-8-LI fibers occurred in the ventromedial region o f the caudal and intermediate AP (Figs. 1D-E; 3A and B). At more rostral level (Fig. 1F) this dense pattern was replaced with scattered to moderate accumulations of CCK-8-LI fibers. The remainder of the caudal AP consisted of a patchwork of scattered to dense CCK-8-LI fibers. At a more rostral level the majority o f the AP was occupied by scattered amounts of CCK-8-LI fibers although moderate accumulation of CCK-8-LI fibers could be found near the ventral and lateral AP borders. At intermediate levels a 'band' of increased immunoreactivity was observed that originated in the ventromedial AP region near the borders of the NTS and D M N (Fig. 3C). This 'band' o f CCK-8-LI fibers swept dorsally and laterally through the medial-half of the AP before diminishing.
Discussion This study provides a detailed immunohistochemical description of CCK-8-LI fibers and cell bodies within the rat AP, and the first visualization of CCK-8-LI fibers within the AP of the cat. CCK-8-LI fibers and cell bodies were found to be differentially distributed in the AP of both species. In the rat AP, CCK-8-LI fibers were most abundant at intermediate levels, whereas in the cat AP, the greatest accumulation of CCK-8-LI fibers were present caudally. In both species the majority of CCK-8-LI fibers were located along the borders of the AP. Large numbers of CCK-8-LI cell bodies were present in the AP o f colchicine-treated rats, but were lacking in the AP o f both untreated and colchicine-treated cats. It is possible that the amount of colchicine administered to the cats was not sufficient to reveal CCK-8-LI cell bodies. However, this is unlikely since additional cats treated with colchicine at levels (200 #g/kg body weight) comparable to that given to rats did not have CCK-8-LI cell bodies in the AP (unpublished observations). The results presented here support and expand upon previous studies that have reported CCK-8-LI fibers within the AP of the rat using PAP and immunohistofluorescent techniques [14,15,22]. In the rat AP, CCK-8-LI fibers probably originate from both intrinsic and extrinsic sources. CCK-8-LI AP neurons provide a portion of these fibers, but a number of them may originate from fibers crossing the AP-NTS
4 Fig. 3. (A) Photomicrographof CCK-8-L1fibersin the AP of the cat. The majority ofimmunostaining is located along the ventromedial border (asterisk). x 60. (B) Photomicrograph of the dense band of CCK-8-LI fibersin the ventromedialregion of the AP (arrow). The CCK-8-LI fibersdecreasein numbers in more dorsal and lateral regions, x 145. (C) Photomicrograph of CCK-8-LI fibers in the dorsal region of the AP. A band of immunoreactivity(arrow) sweeps through this area. x 155.
38 border from CCK-8-LI somata lying within the NTS and DMN [14,15,22]. Another possible source for AP CCK-8-LI fibers are from CCK-8-LI afferents from the vagus nerve [18,19]. Because the cat AP lacks CCK-8-LI somata, CCK-8-LI fibers are necessarily of extrinsic origin, probably arising from CCK-8-LI cell bodies that lie within the NTS (unpublished observations), or from CCK-8-LI vagal afferents [18,19]. Recently, new information has been gathered concerning the role of the AP in cardiovascular function [5,6], and CCK-8 may play a part in this role. CCK-8 has been demonstrated to be involved with the cardiovascular and respiratory functions of the NTS [13,15,26]. Because the AP has extensive reciprocal connections with the NTS and DMN [8,9] a portion of the CCK-8-LI fibers observed in these two nuclei may be of AP origin. If this is the case, then AP CCK-8-LI fibers projecting to the NTS and DMN may be involved in the regulation of cardiovascular control. However, partial cardiovascular control may be intrinsic to the AP. CCK-8-LI fibers and cell bodies are found in greatest numbers around the periphery of the AP; a region that is rich in primary vagal afferents [12,17,27]. Additionally, other neuropeptides involved with cardiovascular regulation, such as substance P and enkephalins, are also found within the AP. These AP substance P and enkephalin immunoreactive fiber patterns overlap and are also most numerous around the periphery of the AP [28]. Closely overlapping distributions of substance P and enkephalin immunoreactivities have also been observed in cardiovascular divisions of the NTS [29,30] which receive large numbers of vagal afferent projections [10-12]. Therefore, similar AP neurotransmitter distribution patterns, associated with terminal fields from vagal afferents, suggest that peripheral AP regions may be a site of autonomic control. Although it has been shown by several investigators that CCK-8 acts as a satiety agent [16,17] the locus of this action has not been elucidated. Researchers [31] have demonstrated that gastric vagotomy attenuates the reduced feeding response induced by exogenous CCK-8, suggesting that CCK-8 acts via a vagal influence. However, intracerebroventricular injections of CCK-8 also reduce feeding [16], suggesting a central site of action. In a recent study van der Kooy [32] found that AP lesions in rats attenuate the satiety effects of exogenous CCK-8. Therefore, the AP may be a site where CCK-8 acts to reduce food intake via a CCK-8 induced malaise. This hypothesis takes into account both of the previously observed vagal and central satiety effects of CCK-8. Accordingly, ablation of the AP will result in the loss of CCK-8 vagal afferents [12,18,19,27], eliminating a peripheral CCK-8 effect, as well as destroying AP CCK-8 receptors [20], eliminating a central CCK-8 effect. The immunohistochemical results presented in our study provide a morphological basis for this hypothesis. Our results demonstrate, at least in the rat, that a portion of the CCK-8-LI in the AP is of an intrinsic nature rather than being solely derived from CCK-8-LI vagal afferents. Although the influences of the vagus in CCK-8 dependent satiety can not be ignored, the large number of CCK-8-LI somata and fibers present in the AP indicate that the AP is a likely site where CCK-8 can induce satiety. To what degree these intrinsic CCK-8-LI AP neurons contribute to satiety is presently not known, but should be considered in future satiety experiments that utilize rats as an experimental model.
39
The differential distribution of CCK-8-LI between emetic and non-emetic species may be related to the ability of a species to demonstrate this reflex behavior. Differences in the distribution of other putative neurotransmitters between emetic and non-emetic species, such as FMRFamide, serotonin, enkephalin, and substance P have also been noted [25,28]. However, more emetic and non-emetic species will have to be examined before a trend can be established that correlates putative neurotransmitter distribution with the emetic reflex.
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