Distribution of cholecystokinin binding sites in the North American opossum cerebellum

Journal of Chemical Neuroanatomy 7 (1994) 105-112

Distribution of cholecystokinin binding sites in the North American opossum cerebellum Paul C. Madtes

Jr.a'b,

James S. King *a

aDepartraent of Cell Biology, Neurobiology and Anatomy, The Ohio State University, 333 West lOth Ave., Columbus, OH 43210, USA bDepartment of Biology, Mount Vernon Nazarene College, 800 Martinsburg Rd., Mt. Vernon, OH 43050, USA Accepted 7 March 1994

Abstract

Previous studies in our laboratory have reported on the differential distribution of several neuropeptides, including the octapeptide cholecystokinin (CCKs), in the cerebellar cortex and nuclei of the North American opossum (Didelphis marsupialis virginiana). The present account reports on the distribution of CCKg binding sites as determined from serial autoradiographic images of the cerebellum which were labelled by using [125I]Bolton Hunter sulfated CCK 8. Evidence for the limited presence of CCKs-like immunoreactivity and CCK 8 binding sites in several other species suggests that the distribution of this peptide and its receptor(s) may be species specific. In the opossum, CCKs-like immunoreactivity is present in mossy fiber terminals that distribute throughout the cerebellar cortex; it has a very limited distribution in climbing fibers (King and Bishop (1990) J. Comp. Neurol. 238, 373-384. CCK8 binding sites are present throughout all lobules of the cerebellar cortex and the cerebellar nuclei, which correlates well with the distribution of the peptide. CCK8-1ike immunoreactivity is located primarily in the granule cell layer, although the greatest density of binding sites is in the molecular layer. The presence of CCK8 in mossy fiber terminals, coupled with the presence of CCK 8 binding sites in the cerebellar cortex, and the fact that CCK 8 alters the firing rate of Purkinje cells (Madtes et al. (1992) Neurosci. Abstr. 18, 853) indicate this peptide may function as a neuromodulator in the cerebellum of the North American opossum. Moreover, the primary distribution of CCKs binding sites in the molecular layer when compared to the distribution of CCK8 in axon terminals in the granule cell layer suggest the action of CCK 8 could be mediated through volume transmission. Keywords: CCK binding site; Mossy fibres; Peptide

1. Introduction

It is well documented that cholecystokinin is present in the brain of many species (Jansen and Lamers, 1985; Crawley, 1985). However, several reports indicate this peptide is either present in very low levels or not present in the adult cerebellum of the human, nonhuman primate, cow, pig, mouse, rat, hamster, Brazilian opossum or guinea pig (Rehfeld, 1978; Larsson and Rehfeld, 1979; Emson et al., 1982; Beinfeld et al., 1983; Beinfeld and Palkovits, 1984; Williams et al., 1986; Fox et al., 1991). In contrast, the analyses of binding sites for * Corresponding author. 0891-0618/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0891-0618(94)00009-S

CCK in the cerebellum have shown striking differences between species. One of the highest number of CCK binding sites in the guinea pig central nervous system has been reported in the cerebellum (Zarbin et al., 1983; Williams et al., 1986). CCK binding sites also are present in the Brazilian opossum cerebellum (KuehlKovarik et al., 1992) and in the human cerebellum within the granule cell layer (Dietl et al., 1987), but are absent from the cerebellum of the rat and hamster (Williams et al., 1986). Williams et al. (1986) found no CCK binding sites in the mouse cerebellum whereas Sekiguchi and Moroji (1986) reported moderate levels. Although CCK8 apparently is involved in neurotransmission, the presence of other forms (e.g., CCK58,

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procholecystokinin, preprocholecystokinin) suggest each could play a functional role in the central nervous system (Eng et al., 1989). The way cells process CCK into different forms may contribute to the variations in the distribution of CCK in different species. In the North American opossum, numerous CCK8like immunoreactive mossy fiber terminals, whose cell bodies of origin are in the brainstem, are present throughout the cerebellar cortex (King and Bishop, 1990). CCKa-like immunoreactivity also is expressed in the same species early in development and has been useful as a marker to define the temporal sequence of mossy fiber development (King and Bishop, 1992). In the present account, we report the distribution of CCK8 binding sites in the adult North American opossum cerebellum and correlate these data with our previous description of the distribution of CCKs-like immunoreactive terminals (King and Bishop, 1990). 2. Materials and methods

2.1. Preparation of tissue Adult opossums (Didelphis marsupialis virginiana) obtained from the wild were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg). A craniotomy was performed to maximally expose the brain, and the brainstem was severed. The cerebellum was removed and hemisected at the mid-line. Each half was frozen in liquid nitrogen and dry ice, and stored at -70°C. Thirty-micron frozen sections were cut on a cryostat (Bright Instruments), thaw-mounted on gelatinized slides, and stored at -70°C until processed for ligand binding. For autoradiography, one half of the cerebellum was processed in the sagittal plane and the other half in the transverse plane. 2.2. Ligand binding of tissue The binding protocol for the preliminary studies was based on previously published studies in the rat (Akesson et al., 1987). Tissue sections were preincubated in 50 mM Tris-HCl buffer, pH 7.7, containing 7.5 mM MgCI 2 and 0.2% bovine serum albumin, for 15 rain at room temperature to remove endogenous CCK. Sections then were incubated for 2 h at room temperature in 50 mM Tris-HCl buffer, pH 7.7, containing 7.5 mM MgCl2, 0.2% bovine serum albumin, 0.02% bacitracin, 1.0 mM dithiothreitol and 0.1 nM [125IlBolton Hunter sulfated CCKs (2200 Ci/mmol, New England Nuclear) to determine total binding. Nonspecific binding was determined in adjacent sections by adding 1 #M unlabelled displacer (CCKs sulfate) to the incubation buffer. Tris-HCl, bovine serum albumin, baeitracin and dithiothreitol were obtained from Sigma

Chemical Company. Unlabelled CCK8 sulfate was obtained from Bachem. After incubation, the tissue sections were washed in phosphate-buffered saline containing 0.01% Triton X-100 for 10 rain (five 2-rain washes) at 4°C and dipped in deionized water at 4°C to remove salts and buffer. Specific binding is defined for both the biochemical and the autoradiographic measurements as the difference between total binding and nonspecific binding. The specific binding ranged from 40 to 50% of the total binding. 2.3. Analysis of ligand binding on tissue sections Following the final wash described in the receptor binding protocol, sections intended for determination of in vitro binding characteristics were wiped from each slide with a glass fiber filter disc (Whatman GF/A), placed individually in a glass tube, and counted in a gamma counter (Beckman 9000; efficiency = 80%). In order to confirm that CCK binding sites in the opossum cerebellum have the same characteristics as those reported for other species, studies were carried out to determine the apparent affinity (KD) of the radiolabelled ligand, [125I]CCKs; the maximum number of binding sites present (Bmax); the time and temperature of pre-incubation, incubation and post-incubation for optimum binding; and the inhibitory constant (K0 of the displacer. The results of these studies also were used as the optimal binding conditions for the autoradiographic labelling studies. In order to determine the KD and Bmax, 7 concentrations of ligand centered around the reported K D of 0.1 nM were employed (Zarbin et a1.,1983; Akesson et al., 1987; Dietl et al., 1987; Goldman et al., 1987; Kritzer et al., 1987; Woodruff et al., 1991). Scatchard analysis was performed using linear regression to obtain the KD and the Bmax. Each point was analysed in triplicate and was repeated in three separate experiments. 2.4. Qualitative autoradiography of tissue Three adult cerebella were analysed for localization of CCK 8 binding sites. Following the final wash described in the receptor binding protocol, slides intended for autoradiography were dried rapidly under a stream of cold, dry air. Care was taken to dry the sections thoroughly in order to prevent diffusion of the reversibly bound radioligand. Subsequent to in vitro labelling of receptors, the slide-mounted sections were screened by apposing slides to X-ray film (Kodak X-OMAT AR) in light-proof containers. Internal 125I-brain paste standards were exposed concurrently with the experimental sections. The films were processed after either two (T 0, three (T2), or four (T3) days of exposure at 4°C. They

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were developed in D-19 (Kodak) for 4 min, rinsed in water for 30 s, fixed in Rapid Fix (Kodak) for 5 min, and rinsed in water for 30 min, before drying. Autoradiographic images also were generated by apposing and gluing coverslips dipped in NTB3 nuclear emulsion (diluted 1:1 with water; Kodak). The slides

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were stored at 4°C until processed for photographic development. The times of exposure and the development process were the same as for the films, except the Rapid Fix was diluted 1:1 with water. After coverslips were developed, all tissue sections were fixed in 4% paraformaldehyde for 30 min. Alternate pairs of tissue sections

Figs. i-4. Fig. 1. is a darkfleld photomicrograph of an emulsion coated slide that illustrates CCK s binding in a sagittal section of the cerebellum; lobules I through X are labelled. An adjacent section (not illustrated) was lightly counterstained for Nissl substance and indicates the greatest density of CCK s binding sites is in the molecular layer. This laminar pattern of distribution is illustrated in a higher magnification photograph of Iobule X (Fig. 2). Figs. 3 and 4 are bright field photomicrographs that illustrate the distribution of CCK s immunoreactive mossy fibers (arrows) in Iobule X. When Fig. 2 is compared to Fig. 4, it is apparent there is a mismatch between the greatest density of receptor binding in the molecular layer (ML, Fig. 2) and the localization of CCK s immunoreactive terminals in the internal granule cell layer (GL, Fig. 4). White matter (WM). The calibration bar in Fig. I = 1.5 mm; in Fig. 3 = 20 #m; in Figs. 2 and 4 = 500 ~m.

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were counter-stained using a Nissl stain. All sections were dehydrated in increasing concentrations of ethanol, cleared in xylene, and coverslipped with Permount. 2.5. Immunohistochemistry

Details of tissue processing and the antibody employed are given in King and Bishop (1990). Characterization of the antibody, which was generated in a New Zealand white rabbit (#182) immunized with sulfated CCK8 conjugated with keyhole limpet hemocyanin, has been published (Sasek et al., 1984). The antisera was a gift from Dr. Robert Elde, University of Minnesota. 3. Results

3.2. Laminar distribution

CCK8 binding sites are present throughout the cerebellum with the greatest density present in the molecular layer (Fig. 1). The distribution in the molecular layer is illustrated at higher magnification in lobule X (Fig. 2). In contrast, CCKs-like immunoreactivity is present in mossy fiber terminals throughout the granule cell layer (Figs. 3 and 4). When the greatest density of silver grains is compared with the distribution of CCKs-like immunoreactive terminals, it is clear that there is a mismatch between the CCKs binding sites which are greatest in the molecular layer (Fig. 2), and the location of CCKrlike immunoreactive mossy fiber terminals visualized in the internal granular layer (Fig. 4). For a detailed account of the distribution of CCKs-like immunoreactive elements within the opossum cerebellum, see King and Bishop (1990).

3.1. Biochemistry

Scatchard analysis of the data revealed that the KD was 0.1 nM and the Bmax was 2.36 fmol/g tissue. The KI for sulfated CCKs (the displacer used in this study) was 10 mM. The optimal specific binding was obtained using a 15 rain pre-incubation at room temperature, a 2-h incubation at room temperature, and five 2-rain postincubation washes at 4°C. Because these values agree with those reported previously (Gaudreau et al., 1983; Akesson et al., 1987; Clark et al., 1986; Dawbarn and Emson, 1984; Dietl et al., 1987; Woodruff et al., 1991), all autoradiographic studies are conducted using these concentrations and conditions to label tissue sections.

3.3. Lobular distribution

The relative density of CCKs binding sites is evident by comparing autoradiograms of total binding (Fig. 5) with an image of nonspecific binding demonstrated in an adjacent section (Fig. 6). In this sagittal section of the vermis (Fig. 5), the density of specific binding is fairly uniform throughout the molecular layer in each of the cerebellar lobules (I-X). When viewed in the transverse plane (Figs. 7-11), the distribution pattern shows some variation between the hemispheres and vermis. Posteriorly, the binding is more intense in the vermis

Figs. 5-6. Fig. 5. is a darkfi¢ld photomicrograph of a film autoradiogram that illustrates total CCK binding in a sagittal section through the vermis. Fig. 6. illustrates nonspecificbinding in a film autoradiogram from an adjacent sagittal section. The calibration bars in Figs. 5 and 6 = 2 ram.

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than the adjacent paramedian lobule (Figs. 7 and 8). In representative anterior sections, the greatest density o f binding is present in crus I compared to lobus simplex and vermal lobules I I - V I (Figs. 10 and I1).

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3.4. Cerebellar nuclei

CCK8 binding sites also are present in all o f the cerebellar nuclei (Fig. 9) which correlates well with the

Figs. 7-11. These are photographs of film autoradiograms to illustrate CCK8 binding sites in transverse sections of the cerebellum. Vermal lobules (II, 111, IV, VI, VII, VIII, IX, X), the paramedian Iobule (PML), the lobus simplex (LS) and crus 1 (CRI) are labelled. The darkfield illustration (Fig. 9) indicates binding in the medial (MN), interpositus (IP) and lateral (LN) cerebellar nuclei. The calibration bar in Fig. 11 = 1 mm and applies to Figs. 7-10.

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distribution of CCKs-like immunoreactive puncta (King and Bishop, 1990). The density of the silver grains is similar in all the cerebeUar nuclei and is comparable to the caudal vermis (VII-X) of the cerebellar cortex. 4. Discussion

The present data provide the first account where the presence of CCK8 receptor binding sites can be correlated with the presence of CCKs-like immunoreactive terminals (King and Bishop, 1990) in the cerebellum. However, the primary laminar distribution of CCK8 binding sites, in the molecular layer, when compared to the distribution of CCKs-like immunoreactive axon terminals in the granule cell layer, provides evidence of a mismatch between terminals which contain the peptide and cells on which the binding sites are located. These morphological observations suggest that volume transmission (Fuxe and Agnati, 1991) could be the mechanism by which CCK influences its target neurons in the opossum cerebellum. As proposed by these authors, a specific neuroactive compound may act, by virtue of its receptor location, at some distance from the site of its release from a nerve terminal. If CCK receptors are present in the molecular layer of the opossum, the location of CCKs-like immunoreactivity in the granule cell layer suggests a paracrine mechanism of action. The effect would be mediated over a distance of between 200 and 400 #m depending on the release site within the granule cell layer, and the location of the proposed CCK receptor. Extracellular recordings of Purkinje cell activity in this species, following the iontophoretic application of CCKs, indicate that this peptide increases the frequency of simple spike activity (Madtes et al., 1992). Taken together, these data support our contention that CCK functions as a neuromodulator in the cerebeUar cortex of the North American opossum. However, in the cerebellar nuclei, CCK8 immunoreactive puncta are present (King et al., 1992) as are CCK 8 binding sites (present results). Thus, the action of CCK8 in the cerebellar nuclei, its mechanism of action, or both, may not be the same as in the cortex. High levels of CCKs-like immunoreactivity have not been reported in the cerebellum of any other species. (See King and Bishop (1990) for a review of this literature.) However, recent experiments in our laboratory using the same antibody reveal CCKs-like immunoreactivity in the Brazilian opossum; the immunoreactive mossy fiber terminals in this species are restricted primarily to lobule IX. In addition, a plexus of beaded axons is located around Purkinje cell bodies (King, unpublished observations). CCK8 binding sites have been reported in the cerebellum of several species including the Brazilian opossum (Kuehl-Kovarik at al., 1993), the guinea pig (Sekiguchi and Moroji, 1986), the human (Dietl at al., 1987; Goldman et al., 1987) and the North

American opossum (present results). In the latter species, the primary distribution is in the molecular layer, but the cellular localization of the receptor has not been determined. Two types of CCK receptors have been identified. One, type A, is found primarily though not exclusively in the periphery, and the second, type B, is localized to the central nervous system (Hill at al., 1992). The mechanism of action for CCK in the cerebellum is not known; however, in the ventromedial nucleus of the hypothalamus, CCK acts through the type B receptor to inactivate an outward potassium conductance resulting in depolarization of the neuron. In the interfascicular nucleus of the hypothalamus, CCK acts through the type A receptor to excite neurons (Hill and Boden, 1989). Several studies have suggested that changes in conductance produced by CCK likely involve second messenger systems (see Kelly and Larkman, 1989). Studies are currently in progress to define the receptor types in the North American opossum cerebellum, and its mechanism of action. The role of CCK in the central nervous system, particularly in the cerebellum, has not been fully elucidated. The species differences confound the issue and make the solution(s) far from simple. It is clear that immunohistochemical and in situ hybridization techniques identify specific molecular forms by labelling specific sites on particular molecules. Recent studies have revealed additional differences between species regarding the forms of CCK and their localization. In addition to the mature forms (CCK58, CCK33, and CCKs) found in the intestine and the nervous system, two other forms have been described. The first, preprocholecystokinin (preproCCK), contains: (a) a signal peptide; (b) an amino terminal flanking peptide; (c) the CCK58 and CCK8 residues; (d) an amidation region; and (e) a carboxyl terminal flanking peptide (Reeve et al., 1989). The second, procholecystokinin (proCCK), has had the signal peptide cleaved (Reeve et al., 1989). Whereas the mature peptides CCK58 and CCK8 have been reported in a limited number of species, preproCCK and proCCK have a wider distribution. In fact, several cells which have preproCCK or proCCK do not contain the mature forms (Eng et al., 1989; Mogensen and Rehfeld, 1989; Rehfeld, 1989; Rehfeld et al., 1992). Apparently, many cells express CCK genes, but do not cleave the signal peptide and/or flanking residues. The apparent lack of completion of peptide processing, called posttranslational attenuation, may be due to an absence or limited activity of the enzyme(s) responsible for cleaving the complete peptide to produce the mature forms. Therefore, one possibility for the apparent variation in CCK localization could be related to the technical approach used to identify the form of CCK present. Immunohistochemical approaches are directed toward the aminated end of the mature peptide. If the cerebella of

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several species do not cleave preproCCK to produce that site, the antibody would not recognize the protein, producing a negative result. Therefore, antibody recognition of the unprocessed forms of CCK may reveal less species variation than previously reported. Furthermore, differences in processing also appear to be present for substance P and somatostatin (Oertel, 1993). Thus, these findings suggest a second possibility: the variation in the forms of CCK and its function, may reflect a fundamental property of several peptides in the CNS. Taken together, the many forms of CCK and two types of CCK receptors must be considered when attempting to interpret the function of CCK in the central nervous system. Reported differences in the distribution of the peptide and its receptor(s) may reflect technological approaches which only label specific molecules, rather than the multiple molecular forms which may be present in a particular area of the central nervous system. Conversely, the differences may reflect a fundamental property of several peptides in the CNS. The resolution of these differences between species awaits more complete characterization of the peptide forms and receptor types described to date. Acknowledgement The authors wish to thank Barbara Diener-Phelan and Yi Fei Chen for assistance with photography and Katharine Dillingham for typing the manuscript. This work was supported by N.I.H. NS-08798. References Akesson, T.R., Mantyh, P.W., Mantyh, C.R., Watt, D.W. and Mieevych, P.E. (1987) Estrous cyclicity of 1251-cholecystokinin octapeptide binding in the ventromedial hypothalamic nucleus. Neuroendocrinology 45, 257-262. Beinfeld, M.C., Lewis, M.E., Eiden, L.E., Nilaver, G. and Pert, C.B. (1983) The distribution of cholecystokinin and vasoactive intestinal peptide in Rhesus monkey brain as determined by radioimmunoassay. Neuropeptides 3, 337-334. Beinfeld, M.C. and Palkovits, M. (1984) Our current understanding of the distribution and neuronal connections of cholecystokinin in the central nervous system. In Cholecystokinin ( CCK) in the Nervous system (eds de Belleroche, J. and Dockray, G.J.), pp. 42-58. Verlag Chemie, Weinham and Ellis Horwood Ltd, Chichester, England. Clark, C.R., Daum, P. and Hughes, J. (1986) A study of the cerebral cortex cholecystokinin receptor using two radiolabelled probes: evidence for a common CCK 8 and CCK 4 cholecystokinin receptor binding site. J. Neurochem. 46, 1094-1101. Crawley, J.N. (1985) Comparative distribution of cholecystokinin and other neuropeptides. Ann. N Y Acad. ScL 448, 1-8. Dawbarn, D. and Emson, P.C. (1984)Cholecystokinin in neurological disease. In Choieeystokinin (CCK) in the Nervous System (eds de Belleroche, J. and Dockray, G.J.), pp. 59-81. Verlag Chemie, Weinham and Ellis Horwood Ltd., Chichester, England. Dietl, M.M., Probst, A. and Palacios, J.M. (1987) On the distribution of cholecystokinin receptor binding sites in the human brain: An autoradiographic study. Synapse I, 169-183.

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Rehfeld, J.F., Mogensen, N.W., Bardram, L., Hilsted, L. and Monstein, H.J. (1992) Expression, but failing maturation of procholecystokinin in cerebellum. Brain Res. 576, 111-119. Sasck, C.A., Seybold, V.S. and Elde, R.P. (1984) The immunohistochemical localization of nine pcptides in the sacral parasympathetic nucleus and the dorsal gray commissure in rat spinal cord. Neuroscience 12, 855-873. Sekiguchi, R. and Moroji, T. (1986) A comparative study on characterization and distribution of cholecystokinin binding sites among the rat, mouse and guinea pig brain. Brain Res. 399, 271-281. Williams, J.A., Gryson, K.A. and McChesney, D.J. (1986) Brain CCK receptors: Species differences in regional distribution and selectivity. Peptides 7, 293-296. Woodruff, G.N., Hill, D.R., Boden, P., Pinnock, R., Singh, L. and Hughes, J. (1991) Functional role of brain CCK receptors. Neuropeptides (Suppl) 19, 45-56. Zarbin, M.A., Innis, R.B, Wamsley, J.K, Snyder, S.H. and Kuhar, M.J. 0983) Autoradiographic localization of cholecystokinin receptors in rodent brain. J. Neurosci. 3, 877-906.