Influence of cholecystokinin on central monoaminergic pathways

Regulatory Peptides, 6 (1983) 99-109

99

Elsevier

Influence of cholecystokinin on central monoaminergic pathways Erik Widerl6v ~.2.., Peter W. Kalivas l, Mark H. Lewis 1, Arthur J. Prange, Jr. 1 and George R. Breese 1 J Department of Psychiatry and Pharmacology, Biological Sciences Research Center, University of North Carolina, School of Medicine, Chapel Hill, NC 27514, U.S.A., and : Psychiatric Research Center, University of Uppsala, Ullerhker Hospital, S- 750 17 Uppsala, Sweden

(Received 3 June 1982; accepted for publication 7 March 1983)

Summa~ Dopamine (DA) and cholecystokinin octapeptide carboxy-terminal (CCK-8) have been found to coexist in some mesolimbic neurons. The present investigation was undertaken in order to study the biochemical and behavioral interactions between CCK-8 and some central monoaminergic pathways. The action of the sulfated form of CCK-8 (10 /~g/10 ~1 intracerebroventricularly) on DA turnover in nucleus accumbens, olfactory tubercles and corpus striatum of the rat was determined after DA synthesis inhibition with a-methyl-p-tyrosine (250 m g / k g i.p.). Also, CCK-8 action (1-30 /~g intracisternally) on DA synthesis was assessed by measuring accumulation of dihydroxyphenylalanine (DOPA) after DOPA-decarboxylase inhibition with NSD- 1015 (m-hydroxybenzylhydrazine, 100 m g / k g i.p.). The contents of DA and its main metabolites, dihydroxyphenylacetic acid and homovanillic acid, together with serotonin and its main metabolite, 5-hydroxyindoleacetic acid (5HIAA), were measured in different brain areas after direct injection of CCK-8 into the ventral tegmental area (A10) or nucleus accumbens. Further, the effect of CCK-8 on amphetamine-induced locomotion and apomorphine-induced stereotypies was studied along with changes in spontaneous locomotion and rearing after CCK-8 injection into the ventral tegmental area and nucleus accumbens. No consistent statistically significant effects of CCK-8 on biochemical or behavioral assessments on measures of DA function were observed. However, injection of high doses of CCK-8 into the ventral tegmental area significantly decreased levels of 5-HIAA in the nucleus accumbens, olfactory tubercles and striatum.

* Address correspondence to: Dr. Erik WiderlOv, Psychiatric Research Center, University of Uppsala,

Uller~tker Hospital, S-750 17 Uppsala, Sweden. 0167-0115/83/$03.00 © 1983 Elsevier Science Publishers B.V.

100 behavior; dopamine; high-performance liquid chromatography (HPLC); interaction; metabolites; neuropeptides; serotonin; site injections

Introduction

Cholecystokinin was first isolated and sequenced as a 33-amino acid intestinal hormone which was found to have cholecystokinetic and pancreozyminic activities [ 1]. Dockray [2] found cholecystokinin-like immunoreactivity in cerebral tissue from different species, a finding later confirmed by others [3-7]. The demonstration of in vivo biosynthesis of cholecystokinin in rat cerebral cortex [8] and the characterization of distinct receptors for cholecystokinin in brain tissue [9-11] provided further evidence for a possible neurotransmitter role for this peptide. Larsson and Rehfeld [6] have demonstrated that cholecystokinin-like immunoreactivity in guinea pig brain consists of several components with different molecular size. The biologically active octapeptide carboxy-terminal fragment of cholecystokinin (CCK-8) seems to represent the most predominant molecular species in brain [12,13]. The physiological role of cholecystokinin is not clear, but it has been suggested to be involved in satiety regulation [ 14,15] and pain perception [16]. Interestingly, CCK-8 has been shown to coexist with dopamine in some mesolimbic neurons [17]. Further, CCK-8 has been reported to interfere with central dopaminergic functions [18,19]. Based on these observations, additional studies were undertaken to examine further the biochemical and behavioral interactions between CCK-8 and brain dopamine-containing neurons.

Materials and Methods General

Male Sprague-Dawley rats (Charles River Breeding Lab., Wilmington, Mass., U.S.A.) were housed 3-4 per cage in a temperature-controlled room with a 12:12 hour light/dark cycle (light on at 6.00 a.m.) with free access to laboratory chow and tap water. CCK-8 was administered to unanaesthetized, manually restrained animals (150-200 g) either intracisternally (IC) in a volume of 25 ~1 or by direct injection (10 #1) into the right lateral ventricle (intracerebroventricular; 1CV) according to the method described by Popick [20]. In this latter method, injections were made with the aid of a rigid sleeve which was positioned over the right lateral ventricle. The method in our hands has been shown to be rapid, accurate and reproducible [21]. Another group of rats (300-400 g) receiving site injections into the A10 area or nucleus accumbens were anaesthetized with 45 m g / k g of pentobarbital and stereotaxically implanted with bilateral 26 ga stainless-steel guide cannulae [22] in the ventral tegmental area or nucleus accumbens. The stereotaxic coordinates used were according to the atlas of Pellegrino et al. [23]. Cannulae placements in the ventral

101

tegmental area were confirmed via light microscopic examination of cresyl violetstained brain slices [22]. The cannulae placements in the nucleus accumbens were verified by macroscopic examination of the cannula track before the final dissection into different brain areas (striata, olfactory tubercles and nuclei accumbens). The animals were first used in behavioral experiments and subsequently in biochemical studies, each animal receiving a maximum of 4 microinjections. CCK-8 or distilled H20 vehicle was injected into the A10 (0.6 ~tl / side/ 30 s) or nucleus accumbens (1.0 /~l / side/ 30 s) of the unrestrained rat with a Sage infusion pump [22]. Synthesis of brain catecholamines was estimated by measuring the accumulation of dihydroxyphenylalanine (DOPA) at 30 min following 100 m g / k g (i.p.) of NSD-1015 to inhibit DOPA decarboxylase [24]. The dose of NSD-1015 has been demonstrated to almost completely block the enzyme giving a linear increase in DOPA accumulation over 30 min [24]. The NSD-1015 was administered 10 min after intracisternal injection of CCK-8 (1, 3, 10 or 30/~g) or saline. 30 min later the animals were killed. The turnover of dopamine in different brain areas was assessed after inhibition of the rate limiting enzyme of catecholamine synthesis, tyrosine hydroxylase, by a-methyl-p-tyrosine (250 m g / k g i.p.). This method has been described in detail elsewhere [25-27]. For all experiments, the animals were killed by decapitation and the brain rapidly removed and placed on an ice-cooled glass plate to allow dissection of brain areas (striatum, olfactory tubercles, nucleus accumbens, frontal cortex, and hypothalamus). After weighing, the brain parts were immediately frozen on dry ice in nalgene tubes (Denville Scientific, Inc., Denville, N J, U.S.A.) and stored at - 80°C until dopamine, serotonin and their metabolites were assayed. Biochemical assays

Dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxy-4-hydroxyphenylacetic acid (HVA), serotonin (5-hydroxytryptamine; 5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were determined in various brain regions utilizing the high-performance liquid chromatography (HPLC) procedure described by Kilts et al. [28]. An HPLC procedure was also used to assay DOPA simultaneously with dopamine after inhibition of DOPA-decarboxylase with NSD-1015 (100 mg/kg). This method has been described in detail elsewhere [29]. Behavioral measurements

Amphetamine-induced locomotor activity was measured in 'doughnut'-shaped activity cages housed singly in sound-attenuated slightly illuminated chambers [30]. In each cage, six photocell sensors were equally spaced around the circular runway. The frequency of interruption of the light beams was automatically recorded every 15 min for a period of up to 3 h following injection. The animals were allowed to habituate in the activity cage for 1 h prior to drug administration, d-Amphetamine sulfate (1 m g / k g i.p.) or saline was given immediately following the ICV injection of CCK-8 (5 or 25/~g in 10 ~tl) or saline (10/~1). Locomotion and rearing behaviors were estimated with a photocell apparatus designed in our laboratory. The cages were built with opaque plexiglass forming a rectangle with the following dimensions: floor 18 cm x 90 cm, height 50 cm. To

102

quantify rearing, a single photocell was placed at the midline of the long axis, 13 cm from the cage floor, and to quantify locomotion a single photocell was placed at the midline of the short axis 4.5 cm from the cage floor. Studies were conducted to evaluate the photocell apparatus. Microinjection of dopamine into the nucleus accumbens produced a dose-related (5.0-40.0 /*g/accumbens) increase in both locomotor and rearing activity counts. Further, this dopamine-induced behavioral hyperactivity was blocked by simultaneous intra-accumbens injection of fluphenazine. In the present study, animals were adapted to the apparatus for 90 rain, microinjected into specific sites with either CCK-8 or distilled water, and immediately returned to the same photocell cage. Locomotor and rearing counts were measured over 10 min intervals for 120 min. At least 72 h later, animals were retested with the complementary injectate, thus each animal received one control injection and a maximum of two microinjections with different doses of CCK-8. Stereotyped behavior was measured using an observational method [21]. Animals were allowed to habituate to the observation cage for 1 h before receiving a drug treatment. Apomorphine (3 m g / k g i.p.) or saline was administered immediately after direct ICV injection of CCK-8 (5 or 25/~g/10 ~1) or saline (10 ~1). Each animal was observed for a 1 min period every 5 min. The 1 min observation period was divided into four 15-s intervals. At the end of each 15-s interval the observer entered the codes for all behaviors (e.g. continuous sniffing, licking, gnawing, inactivity, etc.) that the animal exhibited during that interval. Session length was 45 min in duration and the observers were in all cases blind to the nature of the treatment received by each animal. Chemicals The sulphated form of cholecystokinin COOH terminal octapeptide (CCK-8; SQ 19,844; kindly provided by Mr. S.J. Lucania, E.R. Squibb and Sons, Inc., Princeton, N J, U.S.A.); m-hydroxybenzylhydrazine, di-HC1 (NSD- 1015) and D-amphetamine sulfate (Sigma Chemical Co., St. Louis, MO, U.S.A.); apomorphine HC1 (Merck and Co., Inc., Rahway, N J, U.S.A.), and DL-c~-methyl-p-tyrosine methylester. HC1 (aMT; Aldrich Chemical Co., Inc., Milwaukee, WI, U.S.A.). The administered doses were calculated as the free bases. Statistical methods Means and standard errors of the mean were calculated using standard statistical procedures. Student's t-test was used for statistical comparison between two independent groups. A paired Student's t-statistic was used for the behavioral site-injection experiments. To compare several treated groups with a control group, data were subjected to analysis of variance and tested with Dunnett's t-test [31]. Comparison between several treatment groups was made with Newman-Keul's test [32] following analysis of variance. Locomotor activity was tested using a repeated measures analysis of variance. P-values < 0.05 were considered to be statistically significant. Stereotypy scores were computed as follows: For each 1 min observation period the percentage of intervals in which a specific behavior (e.g. continuous sniffing) occurred was computed for each animal. The mean percent of intervals during which

103

each behavior occurred was computed by treatment condition for the entire session and presented graphically.

Results

Biochemical experiments The action of CCK-8 on the synthesis of brain catecholamines was assessed by measuring the accumulation of DOPA after inhibition of DOPA-decarboxylase with NSD-1015. Groups of animals were treated with saline or various doses (1-30/~g) of CCK-8 intracisternally. Table I shows that no statistically significant differences in DOPA were obtained after CCK treatment in any of the brain areas studied and the content of dopamine and norepinephrine was similarly not affected (data not shown). The turnover of dopamine in certain brain areas also was assessed after inhibition of tyrosine hydroxylase with a-methyl-p-tyrosine (a-MT). Groups of animals given a - M T (250 m g / k g i.p.) 30 min prior to ICV injection of saline (10/~1) or CCK-8 (10 /~g/10 ~tl) were killed at different time-points after the latter treatments. No difference in the turnover rate between the saline and CCK-8 treated groups was observed in the olfactory tubercles or nucleus accumbens utilizing this procedure (Fig. 1). The ventral tegmental area (A10) of the rat brain is the site of origin for the mesolimbic dopamine neurons, which have their terminal areas in the nucleus accumbens and the olfactory tubercles. After injection of CCK-8 into the A10 area, the only observed change within the dopaminergic system was a slight decrease in striatal HVA at 15 min (Table II). In all the areas studied the concentration of 5-HIAA was decreased after CCK-8 injection into the A10 area. Local injection of CCK-8 (10.2 ~tg/1 #1 per side) into nucleus accumbens did not induce any significant effects at 15 min (data not shown).

TABLE I Influence of CCK-8 on DOPA accumulation after DOPA-decarboxylase inhibition (values are expressed in n g / m g protein as means 4-S.E.M.)

Saline + NSD-1015 CCK-8 1/~g+NSD-1015 CCK-8 3 ~ g + N S D - 1 0 1 5 CCK-8 10 ~ g + N S D - 1 0 1 5 CCK-8 30/~g+ NSD-1015

Striatum DOPA

Olfactory tubercles DOPA

Nucleus accumbens DOPA

21.9 + 1.6 23.1 -t- 1.2 18.9+_2.4 24.6+-2.8 21.1 +-2.3

28.7 ! 3.2 30.2+_5.8 30.3 +- 1.9 29.25:1.6 26.0+-2.5

17.7 +- 3.8 11.8+_3.1 17.7_+ 1.2 16.4+-2.1 19.5+_3.4

C C K or saline was administered intracisternally 10 min prior to NSD-1015 (100 m g / k g i.p.). The animals were decapitated 30 min after the last injection.

104 TABLE II Levels of dopamine, serotonin and their principal metabolites in different brain areas after injection o| CCK-8 into the ventral tegmental area a (values are expressed as percent of saline controls ± S.E.M.; n ~ 5-6) DA

HVA

Time after injection (rain)

Striatum

15 40

85.2_+ 4.1 95.0_+ 8.5

95.7_+ 6.1 86.0_+ 3.8* 101.6-+15.4 98.9_+13.1

Olfactory tubercles

15 40

99.0_+ 14.5 92.9_+ 8.6

96.9_+ 14.0 112.0_+11.9 82.5__+ 6.6 73.6-+ 4.9

Nucleus 15 accumbens 40

DOPAC

5-HT

Brain area

107.9_+ 7.6 108.1_+ 6.3 98.7-+ 9.8 104.6_+11.3 104.3_+20.3 93.8_+23.7

5-HIAA

84.1_+ 3.9** 91.4_+ 8.4

83.9_+ 2.2** 98.4-+ 7.6

85.4_+15.7 79.7_+ 7.0

105.7-+19.4 64.2_+ 3.3***

79.9_+ 9.7 99.4_+ 4.4

92.5_+ 6.4 80.8_+ 3.4***

a Infusion of 1.7 #1 CCK-8 in 0.6 #1 per side during 30 s. * P < 0.05; ** P < 0.02; *** P < 0.01 (two-tailed tests).

Behavioral experiments

When groups of animals (n = 5-7) were given CCK-8 (5 or 25 ~g) or saline 1CV immediately followed by an i.p. injection of D-amphetamine sulfate (1 mg/kg) or saline, CCK-8 did not produce a significant alteration in amphetamine-induced (Fig.

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105

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2) or spontaneous (data not shown) locomotor activity over a 3 h measurement period. Likewise, C C K - 8 (5 or 25 / t g / 1 0 /~1 ICV) did not significantly alter the intensity and duration of various stereotypies (sniffing, gnawing, licking, rearing) produced by i.p. injection of apomorphine (3 m g / k g ) , as shown in Fig. 3. Bilateral injection with vehicle (distilled water) into the V T A produced a short duration increase in locomotor and rearing behavior. This results from the injection procedure, as identical handling of the animal without actually infusing the vehicle produces the same increase. Following injection of C C K - 8 into the VTA, a statistically significant decrease in rearing was observed at 10 min after 10.2 /~g C C K - 8 (Fig. 4). L o c o m o t i o n was attenuated at the same time, however, without reaching statistical significance. N o change in locomotion or rearing was produced after C C K - 8 injection (3.4-10.2/~g) into the nucleus accumbens (data not shown).

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MINUTESAFTER INJECTION

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106

Discussion

In the present study, we have focused our interest on the action of CCK-8 on brain areas rich in dopaminergic neurons, i.e. ventral tegmental area (A 10), olfactory tubercles, nucleus accumbens and striatum. We were unable to demonstrate CCK-8induced changes in biosynthesis of catecholamines in the terminal areas, as assessed by DOPA accumulation after inhibition of DOPA-decarboxylase. Neither did we see any changes in dopamine turnover in nucleus accumbens or olfactory tubercles after synthesis inhibition with a-MT. In a previous study [29], 20 ~g CCK-8 IC did not change the levels of dopamine, DOPAC or HVA in striatum or olfactory tubercles compared to saline treatment. When CCK-8 was injected in the A10 area or nucleus accumbens, virtually no effects were observed in the dopamine containing pathways. The slight decrease in the striatal HVA after A10 injection of CCK-8 seems dubious since dopaminergic cell bodies in this brain area provide few DA-containing terminals to the striatum. Thus, using several lines of evidence, we were not able to reproduce the findings reported by others [18,19] that CCK-8 reduces dopamine turnover in discrete brain areas. There are no apparent reasons for these discrepancies. Differences in rat strains, experimental procedures (e.g. assays and dissection techmques), and sources of CCK-8 may be responsible for the conflicting results. After a high dose of CCK-8 (10.2 #g) injected directly into the A10 a decrease in spontaneous locomotion and rearing was noted. However, the large dose required to produce this effect argues against a specific CCK-8 interaction with mesolimbic dopamine neurons. Further, no effects by CCK-8 on the apomorphine-induced stereotypies or amphetamine-induced locomotion were observed. Therefore, our behavioral experiments also showed only minor interactions between CCK-8 and the dopamine systems. Peripheral injections of CCK-8 have been demonstrated to antagonize methylphenidate-induced stereotypy in mice [33] and apomorphine-induced stereotypy in rats [34]. Further, Nemeroff et al. [35] found that the minimal effective dose of CCK-8 in suppressing stress-induced appetite behavior was smaller after peripheral than central administration. These findings, together with the negative findings after central administration of CCK-8 in the present report, might indicate that CCK-8 is acting via a peripheral rather than central mechanism. However, since it has been demonstrated that small amounts of peptides can cross the blood-brain barrier [36], a central action after peripheral administration cannot be ruled out. Only few reports on the biochemical interactions between CCK-8 and the brain serotonergic mechanisms have been published. Fekete et al. [18] have demonstrated a selective decrease in serotonin turnover in the hypothalamus after ICV injection of CCK-8. Our finding that the levels of 5-HIAA decreased in olfactory tubercles, nucleus accumbens and striatum after relatively low doses of CCK-8 injected directly into the ventral tegmental area also supports a serotonin-CCK-8 interaction. In addition to containing dopaminergic perikarya, the ventral tegmental area also contains a moderate density of serotonergic perikarya [37]. While the terminal fields from these serotonergic neurons have not been determined, it is possible that injection of CCK-8 into the ventral tegmental area may modulate an ascending

107

serotonergic system causing the observed decreases in 5-HIAA. Also supporting a serotonin-CCK interaction is the fact that both compoufids have been recognized as satiety substances [38-39]. In fact, Morley [39] has suggested that the anorectic action of serotonin may involve the release of CCK. Furthermore, L-tryptophan, the serotonin precursor, has been demonstrated to decrease brain CCK levels [40]. In conclusion, our investigations do not demonstrate biochemical or behavioral interaction between centrally administered CCK-8 and central dopaminergic pathways except at very high doses. However, considering the high doses required, the functional significance of these observations is questionable. While a dopamine-CCK-8 interaction was not supported by this study, an effect by CCK-8 on serotonin systems was indicated and deserves further investigation.

Acknowledgements The study has been supported by USPHS grants AA-02334, NS-17509 and MH-33217. E. Widerl6v held a Fogarty International Fellowship (5 F05 TW02928-02) at the time of the study. The authors are grateful to Marcine Garrison and Edna Edwards for excellent technical assistance, and to Lisbeth Juuso for typing the manuscript. Part of these data has previously been presented at the FASEB, 66th annual meeting in New Orleans, 15-23 April 1982 [41].

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