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Amphetamine-induced zif 268 mRNA expression in the medial posterior nucleus accumbens in cholecystokinin-A receptor mutant rats
Neuroscience Letters 281 (2000) 17±20 www.elsevier.com/locate/neulet
Amphetamine-induced zif268 mRNA expression in the medial posterior nucleus accumbens in cholecystokinin-A receptor mutant rats P.D. Shilling, D. Feifel*, J.R. Kelsoe Department of Psychiatry, School of Medicine, University of California, San Diego, La Jolla, CA 92103 - 8620, USA Received 23 September 1999; accepted 9 December 1999
Abstract Converging evidence supports a role for cholecystokinin (CCK) in modulating dopamine (DA)-mediated activity in the rat mesolimbic system. In particular, CCK co-localized with mesolimbic DA cells originating in the ventral tegmental area potentiates DA function in the medial posterior nucleus accumbens (mpNA) through CCK-A receptors. Recently, a strain of rats lacking the CCK-A receptor, Otsuka Long Evans Tokushima Fatty (OLETF), has been discovered making it possible to study the mesolimbic DA regulatory role of CCK-A receptors. Previous studies have shown that OLETF rats are less sensitive to amphetamine (AMPH)-induced behavioral effects compared to controls. To determine if this altered sensitivity is associated with decreased AMPH-induced postsynaptic activation in the mpNA in OLETF rats, we performed the following experiment. OLETF (CCK-A mutants) and Long Evans Tokushima Otsuka (LETO) rats (controls) were given subcutaneous injections of either saline or AMPH (5.0 mg/kg). One hour after injection all animals were sacri®ced and activation of the mpNA was assessed using in situ hybridization with antisense probes for zif268 mRNA. AMPH treatment produced a signi®cant up-regulation of zif268 mRNA expression in both OLETF and LETO rats (P # 0:0002), compared to saline treatment. However, AMPH had almost an identical effect on zif268 mRNA expression in the mpNA in both rat strains suggesting similar postsynaptic neural activation. The signi®cance of this AMPH-induced zif268 mRNA expression in these two rat strains and its relationship to CCK function in the nucleus accumbens are discussed. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Amphetamine; Cholecystokinin-A receptors; Nucleus accumbens; Dopamine; zif268 mRNA; In situ hybridization; Gene expression
Cholecystokinin (CCK), one of the most abundant peptide neurotransmitters in the central nervous system (CNS) [25], has been shown to be co-localized with dopamine (DA) in a large proportion of mesolimbic neurons in rats [8] and humans [17]. Two types of CCK receptors have been discovered, type A (`alimentary') and type B (`brain') [1]. While CCK-B receptors are expressed throughout the CNS, CCK-A receptors exhibit a limited distribution in rat brain in areas including: posterior nucleus accumbens, substantia nigra, ventral tegmental area, area postrema, interpeduncular nucleus and the nucleus tractus solitarious [6,7]. Converging evidence suggests that CCK co-localized * Corresponding author. Department of Psychiatry, UCSD Medical Center, 200 West Arbor Drive, San Diego, CA 921038620, USA. Tel.: 11-619-543-2827: fax: 11-619-543-3738. E-mail address: [email protected] (D. Feifel)
with dopamine (DA) in the mesolimbic cells originating in the ventral tegmental area enhances the DA neuromodulatory effect in the posterior medial nucleus accumbens (mpNA), which represents the terminal ®eld of these cells [1,22]. For example, CCK injected into the mpNA enhanced dopamine agonist-induced activation of adenyl cyclase [20], hyperlocomotion [1,22], intracranial self stimulation [3], conditioned operant responding [16] and disruption of prepulse inhibition (PPI) [5]. Importantly, CCK-A receptors in the mpNA play a significant role in this modulation of DA related mesolimbic function by CCK [1,22]. For example, Crawley [2] reported that infusion of CCK-A antagonists into the mpNA blocked CCK-induced increases in dopamine-induced hyperlocomotion, whereas CCK-B antagonists had no effect. In addition, Marshall et al. [13] have reported that in tissue slices from the posterior NA the CCK-induced increases in DA release are blocked by CCK-A but not CCK-B antagonists. The role
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 79 2- 8
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P.D. Shilling et al. / Neuroscience Letters 281 (2000) 17±20
of endogenous CCK acting at CCK-A brain receptors, in resulting mesolimbic DA transmission has been studied using selective CCK-A antagonists. The results are equivocal [22]. Furthermore, there is evidence for CCK/D2 receptor interactions in CCK/DA co-transmission. In this respect, CCK has been reported to modulate D2 receptor af®nity and this modulation of D2 receptors by CCK is most robust in the area of CCK/dopamine coexistence in the NA [12]. Recently a strain of rats, Otsuka Long Evans Tokushima Fatty (OLETF), that lack the CCK-A receptor due to a naturally occurring genetic mutation was discovered [10]. These rats represent an important model to investigate endogenous CCK-DA interaction. For example, OLETF rats exhibit decreased basal locomotion [10] and less amphetamine (AMPH)-induced disruption of prepulse inhibition compared with normal rats [4]. These data are consistent with decreased mesolimbic DA function due to the loss of pro-dopamine CCK-A receptors in the mpNA. AMPH-induced de®cits in PPI and increased locomotion have been shown to be related to nucleus accumbens DA [11,21]. AMPH injection has also been shown to increase activation of the nucleus accumbens as measured by zif268, [14,19,23] an immediate early gene (IEG) marker of neuronal activation [18]. This AMPH-induced activation has been shown to be particularly prominent in the mpNA [19] and it appears to be mediated through a postsynaptic mechanism. Since CCK has been reported to facilitate the effects of DA in the mpNA through CCK-A receptors and rats lacking CCK-A receptors exhibit decreased AMPH-induced behavioral responses, we hypothesized that activation in the mpNA, as measured by the IEG zif268, would be decreased in rats lacking CCK-A receptors. Two groups of adult OLETF rats (n 5±7) (CCK-A receptor mutants) and two groups of Long Evans Tokushuma Otsuka (LETO) rats, the parental strain of OLETF rats (n 5±7), were used (Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd.). Animals were housed individually and habituated to saline injection for 3 consecutive days. On the following day half of the OLETF and half of LETO rats were challenged with saline (subcutaneous) and the other half with AMPH (5.0 mg/kg). This dose was chosen because it has been reported to be the lowest dose of AMPH that produces a consistent elevation in zif268 mRNA expression [14,23]. One hour after challenge animals were sacri®ced since zif268 mRNA expression, in response to AMPH challenge, has been reported to be the maximal at this time point [14]. Their brains were then quick-frozen in isopentane. Brains were sectioned coronally (20 mm), thaw mounted onto Fisher SuperFrostPlus slides and stored at 2808C. In situ hybridization was performed as described by Shilling [19]. Antisense 35Slabelled RNA probes were transcribed in vitro from a 230 base pair cDNA BglII/EcoRI insert from the zif268 cDNA (kindly provided by J. Milbrandt). A sense RNA probe was used on control sections and no speci®c hybridization signal
was detected. Sections were ®xed and acetylated before hybridization. Treated slides were then incubated with hybridization buffer containing 1 £ 10 7 cpm/ml denatured riboprobe, for 20 h at 538C on a slide warmer. Slides were then treated with RNase A (10 mg/ml) for 20 min, and washed with 0.1 £ SSC at 658C for 30 min (washes contained 1 M dithiothreitol). Slides were apposed to Amersham bMax Hyper®lm along with 14C and/or 35S standards. Quanti®cation of zif268 mRNA was accomplished by computerized analysis of digitized images of the mpNA using NIH image (W. Rosband, NIMH). The mpNA was outlined using a manual drawing program and included: (1) the area previously reported to be responsive to CCKA receptor antagonists [2]; and (2) the high AMPH-induced zif268 signal in this region (Fig. 1). Sections were taken at 1.6±0.70 mm from bregma [15]. Slides from all treatment groups were run in the same experiment. Quanti®cation measured pixels/area of mpNA and background from adjacent white matter was subtracted. In addition, for each animal, four adjacent sections containing each region of interest (n 5±7) were analyzed bilaterally (eight data points) and the mean value was used in the statistical analysis. Data were analyzed by two-way ANOVA and two-tailed t-tests. Baseline mpNA zif268 mRNA expression was low in both OLETF and LETO rats (Fig. 1). In addition, baseline expression of zif268 mRNA was not altered in rats lacking CCK-A receptors. Furthermore, there was a signi®cant main effect of AMPH (d:f: 1; 20, F 85:683, P 0:0001) and post-hoc t-tests revealed that both OLETF (230%) and LETO rats (216%) exhibited signi®cant AMPH-induced elevations of zif268 mRNA expression (P # 0:0002) in the mpNA (Figs. 1 and 2). A similar effect in both strains was observed in the dorsal striatum (data not shown). This zif268 mRNA elevation in the NA was most prominent in
Fig. 1. The effect of AMPH treatment on zif268 mRNA expression in the mpNA. Film autoradiograms visualizing 35S labelled antisense RNA probes hybridized to the nucleus accumbens of LETO rats treated with saline (A) or AMPH (B) and OLETF rats treated with saline (C) or AMPH (D).
P.D. Shilling et al. / Neuroscience Letters 281 (2000) 17±20
Fig. 2. The effect of AMPH treatment on zif268 mRNA expression in the mpNA represented in optical density units (OD). Animals were sacri®ced 1 h after the AMPH challenge. Symbols above bars represent signi®cant differences, *P # 0:0002, two-tailed ttests. Data points represent the mean ^ SEM of ®ve to seven animals/group.
the medial region as was previously reported [19]. Importantly, there was no interaction of strain and drug. This is the ®rst report of the effect of CCK-A receptors on AMPH-induced zif268 mRNA response in the NA. We report here a signi®cant up-regulation of mpNA zif268 mRNA expression in both OLETF and LETO rats 1 h after the AMPH challenge. However, this up-regulation was almost identical in both rat strains suggesting that the lack of CCK-A receptors does not affect the zif268 mRNA response to AMPH challenge in the mpNA. Some of these results (AMPH-induced elevation of zif268 mRNA) support and extend several other reports in the literature. Consistent with these data Moratella et al. [14], Wang and McGinty [23] and Shilling et al. [19] observed an increase in zif268 mRNA expression after acute AMPH challenge. Also consistent with our previous observations, this AMPHinduced up-regulation of zif268 mRNA in the NA was most evident in the medial region [19]. Our hypothesis of a decreased AMPH-induced activation of the mpNA in rats lacking CCK-A receptors was not con®rmed. It is surprising that activation, as measured by zif268 mRNA, in the mpNA was not altered in rats lacking CCK-A receptors since CCK potentiates DA mediated function in this region. However, it is possible that this potentiation of DA-related behaviors through CCK-A receptors is produced by a mechanism that is distinct from one that regulates zif268 mRNA expression. In this respect, zif268 mRNA expression and CCK modulation of DA behaviors could be dissociated. For example, an AMPH dose of 2 mg/kg results in locomotor activation in most rats. However, zif268 mRNA expression is not consistently elevated [23] at this AMPH
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dose. In addition, measures of zif268 mRNA expression may not be sensitive enough to detect CCK-A receptor modulated differences in activation. Another measure of postsynaptic activity might be able to detect this CCK mediated DA potentiation. For example, CCK administration has been reported to increase adenylate cyclase activity in the posterior NA through D1 receptors [20]. If CCK-A receptors are localized postsynaptically in the NA then one might hypothesize that this CCK mediated increase in adenylate cyclase would be blocked in the OLETF rats. Another plausible explanation for the lack of changes in AMPH-induced mpNA mRNA expression in CCK-A mutants is the complex interactions of peripheral and central CCK through CCK-A receptors. For example, in normal rats peripheral CCK inhibits mesolimbic DA function through CCK-A receptors [9] while central CCK potentiates DA related activity in the mpNA [22]. Therefore, in rats lacking CCK-A receptors peripheral action of CCK on midbrain DA neurons would be disinibited with a concomitant centrally mediated inhibition of the midbrain DA/CCK projections to the mpNA. It is possible that these opposing effects on zif268 mRNA negate each other. However, differential AMPH-induced behavioral changes in OLETF and LETO rats would appear to be inconsistent with this explanation [4]. Nevertheless, this explanation would be reconcilable with behavioral changes if these behavioral changes were modulated by circuitry outside the NA [24]. It is also important to note that the dose of AMPH used in this study (5.0 mg/kg) was higher than doses used in previous studies addressing CCK induced changes in DAmediated behaviors. It is entirely possible that CCK has a different effect at higher doses of AMPH. AMPH-induced DA release is dose dependent [11], e.g. more DA is released at higher doses of AMPH. Since the action of CCK is dependent on activating levels of DA, the amount of DA released is likely to in¯uence the modulating effect of CCK on dopaminergic systems. Furthermore, the results of the effect of AMPH on OLETF rats may be related to the distinct modulation by CCK of DA receptor subtypes re¯ected in a developmental compensatory regulation of postsynaptic DA receptors. Since AMPH-induced zif268 mRNA expression is dependent on D1 receptor activation [14] this would suggest that D1 activity is unchanged in OLETF rats and a decrease in mpNA postsynaptic activity might be the result of a developmental compensatory down-regulation of mpNA D2 receptors. Investigation of both D2 mRNA and D2 receptor binding alterations in CCK-A mutants in the mpNA would address this issue. In addition to the previously hypothesized developmental compensation in D2 receptors, it is possible that any number of compensatory changes could have occurred in OLETF rats. In this respect, studying the effects of a CCK-A antagonist on AMPH-induced zif268 mRNA expression in LETO could address some of these alternative possibilities. In conclusion, we observed a signi®cant up-regulation of
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P.D. Shilling et al. / Neuroscience Letters 281 (2000) 17±20
AMPH- induced mpNA zif268 mRNA expression in both OLETF and LETO rats. However, this up-regulation was almost identical in both rat strains suggesting that the lack of CCK-A receptors does not affect the zif268 mRNA response to AMPH challenge in the mpNA. Alternative measures of postsynaptic activation might enable researchers to detect signi®cant differences in activation of the mpNA in rats lacking CCK-A receptors. Further study of these CCK-A mutants is warranted, in that the action of CCK and its receptors in mesolimbic DA pathways suggests a possible role for this peptide and its receptors in various psychiatric disorders, such as schizophrenia, that involve DA dysregulation. P.D.S. was supported in part by 5 T32MH18399 (Fellowship: Clinical Psychopharmacology and Psychobiology) from the National Institute of Mental Health. D.F. is the recipient of a NARSAD grant. We thank Dr. Kazuyuki Kawano and the Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd., Otsuka, Japan for kindly providing OLETF and LETO rats. We also thank Dr. Jeff Milbrandt for kindly supplying the zif268 cDNA clone and Dr. Richard Granger for use of his image analysis system. Thanks also goes to Tami Reza and Stephanie Robeck for excellent technical assistance. [1] Crawley, J.N., Cholecystokinin-dopamine interactions. Trends Pharmacol. Sci., 12 (1991) 232±236. [2] Crawley, J.N., Subtype-selective cholecystokinin receptor antagonists block cholecystokinin modulation of dopamine-mediated behaviors in the rat mesolimbic pathway. J. Neurosci, 12 (1992) 3380±3391. [3] De Witte, P., Heidbreder, C., Roques, C. and Vandehaeghen, J.J., Opposite effects of cholecystokinin octapeptide (CCK-8) and tetrapeptide (CCK-4) after injection into the caudal part of the nucleus accumbens or into its rostral part and the cerebral ventricles. Neurochem. Int., 10 (1987) 473±479. [4] Feifel, D. and Reza, T., Startle differences in rats not expressing CCK-A receptors. Soc. Neurosci. (Abstracts), 24 (1998) 1924. [5] Feifel, D. and Swerdlow, N.R., The modulation of sensorimotor gating de®cits by mesolimbic cholecystokinin. Neurosci. Lett., 229 (1997) 5±8. [6] Graham, W.C., Hill, D.R., Woodruff, G.N., Sambrook, M.A. and Crossman, A.R., Reduction of [ 125I]Bolton Hunter CCK8 and [ 3H]MK-329 (devazepide) binding to CCK receptors in the substantia nigra/VTA complex and its forebrain projection areas following MPTP-induced hemi-parkinsonism in the monkey. Neurosci. Lett., 131 (1991) 129-134. [7] Hill, D.R., Campbell, N.J., Shaw, T.M. and Woodruff, G.N., Autoradiographic localization and biochemical characterization of peripheral type CCK receptors in rat CNS using highly selective non-peptide CCK antagonists. J. Neurosci., 7 (1987) 2967±2976. [8] Hokfelt, T., Rehfeld, J.F., Skirboll, L., Ivemark, B., Goldstein, M. and Markey, K., Evidence for coexistence of dopamine and CCK in meso-limbic neurones. Nature, 285 (1980) 476± 478. [9] Kihara, T., Ikeda, M., Matsubara, K. and Matsushita, A., Differential effects of ceruletide on amphetamine-induced
[10]
[11]
[12]
[13]
[14] [15] [16]
[17]
[18] [19]
[20]
[21]
[22] [23]
[24]
[25]
behaviors and regional dopamine release in the rat. Eur. J. Pharmacol., 230 (1993) 271±277. Kobayashi, S., Ohta, M., Miyasaka, K. and Funakoshi, A., Decrease in exploratory behavior in naturally occurring cholecystokinin (CCK)-A receptor gene knockout rats. Neurosci. Lett., 214 (1996) 61±64. Kuczenski, R., Melega, W.P., Cho, A.K. and Segal, D.S., Extracellular dopamine and amphetamine after systemic amphetamine administration: comparison to the behavioral response. J. Pharmacol. Exp. Ther., 282 (1997) 591± 596. Li, X.M., Hedlund, P.B. and Fuxe, K., Cholecystokinin octapeptide in vitro and ex vivo strongly modulates striatal dopamine D2 receptors in rat forebrain sections. Eur. J. Neurosci., 7 (1995) 962±971. Marshall, F.H., Barnes, S., Hughes, J., Woodruff, G.N. and Hunter, J.C., Cholecystokinin modulates the release of dopamine from the anterior and posterior nucleus accumbens by two different mechanisms. J. Neurochem., 56 (1991) 917±922. Moratalla, R., Robertson, H.A. and Graybiel, A.M., Dynamic regulation of NGFI-A (zif268, egr1) gene expression in the striatum. J. Neurosci., 12 (1992) 2609±2622. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego, CA, 1997. Phillips, G.D., Le Noury, J., Wolterink, G., DonselaarWolterink, I., Robbins, T.W. and Everitt, B.J., Cholecystokinin-dopamine interactions within the nucleus accumbens in the control over behaviour by conditioned reinforcement. Behav. Brain Res., 55 (1993) 223±231. Schalling, M., Friberg, K. and Seroogy, K., Analysis of expression of cholecystokinin in dopamine cells in the ventral mecencephalon of several species and human with schizophrenia. Proc. Natl. Acad. Sci. USA, 87 (1990) 8427±8431. Sheng, M. and Greenberg, M.E., The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron., 4 (1990) 477±485. Shilling, P.D., Kelsoe, J.R., Kuczenski, R. and Segal, D.S., Differential regional zif268 mRNA expression in an escalating dose/binge model of amphetamine-induced psychosis. Neuroscience, 96(1) (2000) 83±90. Studler, J.M., Reibaud, M., Herve, D., Blanc, G., Glowinski, J. and Tassin, J.P., Opposite effects of sulfated cholecystokinin on DA-sensitive adenylate cyclase in two areas of the rat nucleus accumbens. Eur. J. Pharmacol., 126 (1986) 125±128. Swerdlow, N.R., Mansbach, R.S., Geyer, M.A., Pulvirenti, L., Koob, G.F. and Braff, D.L., Amphetamine disruption of prepulse inhibition of acoustic startle is reversed by depletion of mesolimbic dopamine. Psychopharmacology (Berl)., 100 (1990) 413±416. Vaccarino, F.J., Nucleus accumbens dopamine-CCK interactions in psychostimulant reward and related behaviors. Neurosci. Biobehav. Rev., 18 (1994) 207±214. Wang, J.Q., Smith, A.J. and McGinty, J.F., A single injection of amphetamine or methamphetamine induces dynamic alterations in c-fos, zif268 and preprodynorphin messenger RNA expression in rat forebrain. Neuroscience, 68 (1995) 83±95. Wolf, M.E., Dahlin, S.L., Hu, X.T., Xue, C.J. and White, K., Effects of lesions of prefrontal cortex, amygdala, or fornix on behavioral sensitization to amphetamine: comparison with N-methyl-D-aspartate antagonists. Neuroscience, 69 (1995) 417±439. Woodruff, G.N., Hill, D.R., Boden, P., Pinnock, R., Singh, L. and Hughes, J., Functional role of brain CCK receptors. Neuropeptides, 19 (1991) 45±56.
Amphetamine-induced zif268 mRNA expression in the medial posterior nucleus accumbens in cholecystokinin-A receptor mutant rats P.D. Shilling, D. Feifel*, J.R. Kelsoe Department of Psychiatry, School of Medicine, University of California, San Diego, La Jolla, CA 92103 - 8620, USA Received 23 September 1999; accepted 9 December 1999
Abstract Converging evidence supports a role for cholecystokinin (CCK) in modulating dopamine (DA)-mediated activity in the rat mesolimbic system. In particular, CCK co-localized with mesolimbic DA cells originating in the ventral tegmental area potentiates DA function in the medial posterior nucleus accumbens (mpNA) through CCK-A receptors. Recently, a strain of rats lacking the CCK-A receptor, Otsuka Long Evans Tokushima Fatty (OLETF), has been discovered making it possible to study the mesolimbic DA regulatory role of CCK-A receptors. Previous studies have shown that OLETF rats are less sensitive to amphetamine (AMPH)-induced behavioral effects compared to controls. To determine if this altered sensitivity is associated with decreased AMPH-induced postsynaptic activation in the mpNA in OLETF rats, we performed the following experiment. OLETF (CCK-A mutants) and Long Evans Tokushima Otsuka (LETO) rats (controls) were given subcutaneous injections of either saline or AMPH (5.0 mg/kg). One hour after injection all animals were sacri®ced and activation of the mpNA was assessed using in situ hybridization with antisense probes for zif268 mRNA. AMPH treatment produced a signi®cant up-regulation of zif268 mRNA expression in both OLETF and LETO rats (P # 0:0002), compared to saline treatment. However, AMPH had almost an identical effect on zif268 mRNA expression in the mpNA in both rat strains suggesting similar postsynaptic neural activation. The signi®cance of this AMPH-induced zif268 mRNA expression in these two rat strains and its relationship to CCK function in the nucleus accumbens are discussed. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Amphetamine; Cholecystokinin-A receptors; Nucleus accumbens; Dopamine; zif268 mRNA; In situ hybridization; Gene expression
Cholecystokinin (CCK), one of the most abundant peptide neurotransmitters in the central nervous system (CNS) [25], has been shown to be co-localized with dopamine (DA) in a large proportion of mesolimbic neurons in rats [8] and humans [17]. Two types of CCK receptors have been discovered, type A (`alimentary') and type B (`brain') [1]. While CCK-B receptors are expressed throughout the CNS, CCK-A receptors exhibit a limited distribution in rat brain in areas including: posterior nucleus accumbens, substantia nigra, ventral tegmental area, area postrema, interpeduncular nucleus and the nucleus tractus solitarious [6,7]. Converging evidence suggests that CCK co-localized * Corresponding author. Department of Psychiatry, UCSD Medical Center, 200 West Arbor Drive, San Diego, CA 921038620, USA. Tel.: 11-619-543-2827: fax: 11-619-543-3738. E-mail address: [email protected] (D. Feifel)
with dopamine (DA) in the mesolimbic cells originating in the ventral tegmental area enhances the DA neuromodulatory effect in the posterior medial nucleus accumbens (mpNA), which represents the terminal ®eld of these cells [1,22]. For example, CCK injected into the mpNA enhanced dopamine agonist-induced activation of adenyl cyclase [20], hyperlocomotion [1,22], intracranial self stimulation [3], conditioned operant responding [16] and disruption of prepulse inhibition (PPI) [5]. Importantly, CCK-A receptors in the mpNA play a significant role in this modulation of DA related mesolimbic function by CCK [1,22]. For example, Crawley [2] reported that infusion of CCK-A antagonists into the mpNA blocked CCK-induced increases in dopamine-induced hyperlocomotion, whereas CCK-B antagonists had no effect. In addition, Marshall et al. [13] have reported that in tissue slices from the posterior NA the CCK-induced increases in DA release are blocked by CCK-A but not CCK-B antagonists. The role
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 79 2- 8
18
P.D. Shilling et al. / Neuroscience Letters 281 (2000) 17±20
of endogenous CCK acting at CCK-A brain receptors, in resulting mesolimbic DA transmission has been studied using selective CCK-A antagonists. The results are equivocal [22]. Furthermore, there is evidence for CCK/D2 receptor interactions in CCK/DA co-transmission. In this respect, CCK has been reported to modulate D2 receptor af®nity and this modulation of D2 receptors by CCK is most robust in the area of CCK/dopamine coexistence in the NA [12]. Recently a strain of rats, Otsuka Long Evans Tokushima Fatty (OLETF), that lack the CCK-A receptor due to a naturally occurring genetic mutation was discovered [10]. These rats represent an important model to investigate endogenous CCK-DA interaction. For example, OLETF rats exhibit decreased basal locomotion [10] and less amphetamine (AMPH)-induced disruption of prepulse inhibition compared with normal rats [4]. These data are consistent with decreased mesolimbic DA function due to the loss of pro-dopamine CCK-A receptors in the mpNA. AMPH-induced de®cits in PPI and increased locomotion have been shown to be related to nucleus accumbens DA [11,21]. AMPH injection has also been shown to increase activation of the nucleus accumbens as measured by zif268, [14,19,23] an immediate early gene (IEG) marker of neuronal activation [18]. This AMPH-induced activation has been shown to be particularly prominent in the mpNA [19] and it appears to be mediated through a postsynaptic mechanism. Since CCK has been reported to facilitate the effects of DA in the mpNA through CCK-A receptors and rats lacking CCK-A receptors exhibit decreased AMPH-induced behavioral responses, we hypothesized that activation in the mpNA, as measured by the IEG zif268, would be decreased in rats lacking CCK-A receptors. Two groups of adult OLETF rats (n 5±7) (CCK-A receptor mutants) and two groups of Long Evans Tokushuma Otsuka (LETO) rats, the parental strain of OLETF rats (n 5±7), were used (Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd.). Animals were housed individually and habituated to saline injection for 3 consecutive days. On the following day half of the OLETF and half of LETO rats were challenged with saline (subcutaneous) and the other half with AMPH (5.0 mg/kg). This dose was chosen because it has been reported to be the lowest dose of AMPH that produces a consistent elevation in zif268 mRNA expression [14,23]. One hour after challenge animals were sacri®ced since zif268 mRNA expression, in response to AMPH challenge, has been reported to be the maximal at this time point [14]. Their brains were then quick-frozen in isopentane. Brains were sectioned coronally (20 mm), thaw mounted onto Fisher SuperFrostPlus slides and stored at 2808C. In situ hybridization was performed as described by Shilling [19]. Antisense 35Slabelled RNA probes were transcribed in vitro from a 230 base pair cDNA BglII/EcoRI insert from the zif268 cDNA (kindly provided by J. Milbrandt). A sense RNA probe was used on control sections and no speci®c hybridization signal
was detected. Sections were ®xed and acetylated before hybridization. Treated slides were then incubated with hybridization buffer containing 1 £ 10 7 cpm/ml denatured riboprobe, for 20 h at 538C on a slide warmer. Slides were then treated with RNase A (10 mg/ml) for 20 min, and washed with 0.1 £ SSC at 658C for 30 min (washes contained 1 M dithiothreitol). Slides were apposed to Amersham bMax Hyper®lm along with 14C and/or 35S standards. Quanti®cation of zif268 mRNA was accomplished by computerized analysis of digitized images of the mpNA using NIH image (W. Rosband, NIMH). The mpNA was outlined using a manual drawing program and included: (1) the area previously reported to be responsive to CCKA receptor antagonists [2]; and (2) the high AMPH-induced zif268 signal in this region (Fig. 1). Sections were taken at 1.6±0.70 mm from bregma [15]. Slides from all treatment groups were run in the same experiment. Quanti®cation measured pixels/area of mpNA and background from adjacent white matter was subtracted. In addition, for each animal, four adjacent sections containing each region of interest (n 5±7) were analyzed bilaterally (eight data points) and the mean value was used in the statistical analysis. Data were analyzed by two-way ANOVA and two-tailed t-tests. Baseline mpNA zif268 mRNA expression was low in both OLETF and LETO rats (Fig. 1). In addition, baseline expression of zif268 mRNA was not altered in rats lacking CCK-A receptors. Furthermore, there was a signi®cant main effect of AMPH (d:f: 1; 20, F 85:683, P 0:0001) and post-hoc t-tests revealed that both OLETF (230%) and LETO rats (216%) exhibited signi®cant AMPH-induced elevations of zif268 mRNA expression (P # 0:0002) in the mpNA (Figs. 1 and 2). A similar effect in both strains was observed in the dorsal striatum (data not shown). This zif268 mRNA elevation in the NA was most prominent in
Fig. 1. The effect of AMPH treatment on zif268 mRNA expression in the mpNA. Film autoradiograms visualizing 35S labelled antisense RNA probes hybridized to the nucleus accumbens of LETO rats treated with saline (A) or AMPH (B) and OLETF rats treated with saline (C) or AMPH (D).
P.D. Shilling et al. / Neuroscience Letters 281 (2000) 17±20
Fig. 2. The effect of AMPH treatment on zif268 mRNA expression in the mpNA represented in optical density units (OD). Animals were sacri®ced 1 h after the AMPH challenge. Symbols above bars represent signi®cant differences, *P # 0:0002, two-tailed ttests. Data points represent the mean ^ SEM of ®ve to seven animals/group.
the medial region as was previously reported [19]. Importantly, there was no interaction of strain and drug. This is the ®rst report of the effect of CCK-A receptors on AMPH-induced zif268 mRNA response in the NA. We report here a signi®cant up-regulation of mpNA zif268 mRNA expression in both OLETF and LETO rats 1 h after the AMPH challenge. However, this up-regulation was almost identical in both rat strains suggesting that the lack of CCK-A receptors does not affect the zif268 mRNA response to AMPH challenge in the mpNA. Some of these results (AMPH-induced elevation of zif268 mRNA) support and extend several other reports in the literature. Consistent with these data Moratella et al. [14], Wang and McGinty [23] and Shilling et al. [19] observed an increase in zif268 mRNA expression after acute AMPH challenge. Also consistent with our previous observations, this AMPHinduced up-regulation of zif268 mRNA in the NA was most evident in the medial region [19]. Our hypothesis of a decreased AMPH-induced activation of the mpNA in rats lacking CCK-A receptors was not con®rmed. It is surprising that activation, as measured by zif268 mRNA, in the mpNA was not altered in rats lacking CCK-A receptors since CCK potentiates DA mediated function in this region. However, it is possible that this potentiation of DA-related behaviors through CCK-A receptors is produced by a mechanism that is distinct from one that regulates zif268 mRNA expression. In this respect, zif268 mRNA expression and CCK modulation of DA behaviors could be dissociated. For example, an AMPH dose of 2 mg/kg results in locomotor activation in most rats. However, zif268 mRNA expression is not consistently elevated [23] at this AMPH
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dose. In addition, measures of zif268 mRNA expression may not be sensitive enough to detect CCK-A receptor modulated differences in activation. Another measure of postsynaptic activity might be able to detect this CCK mediated DA potentiation. For example, CCK administration has been reported to increase adenylate cyclase activity in the posterior NA through D1 receptors [20]. If CCK-A receptors are localized postsynaptically in the NA then one might hypothesize that this CCK mediated increase in adenylate cyclase would be blocked in the OLETF rats. Another plausible explanation for the lack of changes in AMPH-induced mpNA mRNA expression in CCK-A mutants is the complex interactions of peripheral and central CCK through CCK-A receptors. For example, in normal rats peripheral CCK inhibits mesolimbic DA function through CCK-A receptors [9] while central CCK potentiates DA related activity in the mpNA [22]. Therefore, in rats lacking CCK-A receptors peripheral action of CCK on midbrain DA neurons would be disinibited with a concomitant centrally mediated inhibition of the midbrain DA/CCK projections to the mpNA. It is possible that these opposing effects on zif268 mRNA negate each other. However, differential AMPH-induced behavioral changes in OLETF and LETO rats would appear to be inconsistent with this explanation [4]. Nevertheless, this explanation would be reconcilable with behavioral changes if these behavioral changes were modulated by circuitry outside the NA [24]. It is also important to note that the dose of AMPH used in this study (5.0 mg/kg) was higher than doses used in previous studies addressing CCK induced changes in DAmediated behaviors. It is entirely possible that CCK has a different effect at higher doses of AMPH. AMPH-induced DA release is dose dependent [11], e.g. more DA is released at higher doses of AMPH. Since the action of CCK is dependent on activating levels of DA, the amount of DA released is likely to in¯uence the modulating effect of CCK on dopaminergic systems. Furthermore, the results of the effect of AMPH on OLETF rats may be related to the distinct modulation by CCK of DA receptor subtypes re¯ected in a developmental compensatory regulation of postsynaptic DA receptors. Since AMPH-induced zif268 mRNA expression is dependent on D1 receptor activation [14] this would suggest that D1 activity is unchanged in OLETF rats and a decrease in mpNA postsynaptic activity might be the result of a developmental compensatory down-regulation of mpNA D2 receptors. Investigation of both D2 mRNA and D2 receptor binding alterations in CCK-A mutants in the mpNA would address this issue. In addition to the previously hypothesized developmental compensation in D2 receptors, it is possible that any number of compensatory changes could have occurred in OLETF rats. In this respect, studying the effects of a CCK-A antagonist on AMPH-induced zif268 mRNA expression in LETO could address some of these alternative possibilities. In conclusion, we observed a signi®cant up-regulation of
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AMPH- induced mpNA zif268 mRNA expression in both OLETF and LETO rats. However, this up-regulation was almost identical in both rat strains suggesting that the lack of CCK-A receptors does not affect the zif268 mRNA response to AMPH challenge in the mpNA. Alternative measures of postsynaptic activation might enable researchers to detect signi®cant differences in activation of the mpNA in rats lacking CCK-A receptors. Further study of these CCK-A mutants is warranted, in that the action of CCK and its receptors in mesolimbic DA pathways suggests a possible role for this peptide and its receptors in various psychiatric disorders, such as schizophrenia, that involve DA dysregulation. P.D.S. was supported in part by 5 T32MH18399 (Fellowship: Clinical Psychopharmacology and Psychobiology) from the National Institute of Mental Health. D.F. is the recipient of a NARSAD grant. We thank Dr. Kazuyuki Kawano and the Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd., Otsuka, Japan for kindly providing OLETF and LETO rats. We also thank Dr. Jeff Milbrandt for kindly supplying the zif268 cDNA clone and Dr. Richard Granger for use of his image analysis system. Thanks also goes to Tami Reza and Stephanie Robeck for excellent technical assistance. [1] Crawley, J.N., Cholecystokinin-dopamine interactions. Trends Pharmacol. Sci., 12 (1991) 232±236. [2] Crawley, J.N., Subtype-selective cholecystokinin receptor antagonists block cholecystokinin modulation of dopamine-mediated behaviors in the rat mesolimbic pathway. J. Neurosci, 12 (1992) 3380±3391. [3] De Witte, P., Heidbreder, C., Roques, C. and Vandehaeghen, J.J., Opposite effects of cholecystokinin octapeptide (CCK-8) and tetrapeptide (CCK-4) after injection into the caudal part of the nucleus accumbens or into its rostral part and the cerebral ventricles. Neurochem. Int., 10 (1987) 473±479. [4] Feifel, D. and Reza, T., Startle differences in rats not expressing CCK-A receptors. Soc. Neurosci. (Abstracts), 24 (1998) 1924. [5] Feifel, D. and Swerdlow, N.R., The modulation of sensorimotor gating de®cits by mesolimbic cholecystokinin. Neurosci. Lett., 229 (1997) 5±8. [6] Graham, W.C., Hill, D.R., Woodruff, G.N., Sambrook, M.A. and Crossman, A.R., Reduction of [ 125I]Bolton Hunter CCK8 and [ 3H]MK-329 (devazepide) binding to CCK receptors in the substantia nigra/VTA complex and its forebrain projection areas following MPTP-induced hemi-parkinsonism in the monkey. Neurosci. Lett., 131 (1991) 129-134. [7] Hill, D.R., Campbell, N.J., Shaw, T.M. and Woodruff, G.N., Autoradiographic localization and biochemical characterization of peripheral type CCK receptors in rat CNS using highly selective non-peptide CCK antagonists. J. Neurosci., 7 (1987) 2967±2976. [8] Hokfelt, T., Rehfeld, J.F., Skirboll, L., Ivemark, B., Goldstein, M. and Markey, K., Evidence for coexistence of dopamine and CCK in meso-limbic neurones. Nature, 285 (1980) 476± 478. [9] Kihara, T., Ikeda, M., Matsubara, K. and Matsushita, A., Differential effects of ceruletide on amphetamine-induced
[10]
[11]
[12]
[13]
[14] [15] [16]
[17]
[18] [19]
[20]
[21]
[22] [23]
[24]
[25]
behaviors and regional dopamine release in the rat. Eur. J. Pharmacol., 230 (1993) 271±277. Kobayashi, S., Ohta, M., Miyasaka, K. and Funakoshi, A., Decrease in exploratory behavior in naturally occurring cholecystokinin (CCK)-A receptor gene knockout rats. Neurosci. Lett., 214 (1996) 61±64. Kuczenski, R., Melega, W.P., Cho, A.K. and Segal, D.S., Extracellular dopamine and amphetamine after systemic amphetamine administration: comparison to the behavioral response. J. Pharmacol. Exp. Ther., 282 (1997) 591± 596. Li, X.M., Hedlund, P.B. and Fuxe, K., Cholecystokinin octapeptide in vitro and ex vivo strongly modulates striatal dopamine D2 receptors in rat forebrain sections. Eur. J. Neurosci., 7 (1995) 962±971. Marshall, F.H., Barnes, S., Hughes, J., Woodruff, G.N. and Hunter, J.C., Cholecystokinin modulates the release of dopamine from the anterior and posterior nucleus accumbens by two different mechanisms. J. Neurochem., 56 (1991) 917±922. Moratalla, R., Robertson, H.A. and Graybiel, A.M., Dynamic regulation of NGFI-A (zif268, egr1) gene expression in the striatum. J. Neurosci., 12 (1992) 2609±2622. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego, CA, 1997. Phillips, G.D., Le Noury, J., Wolterink, G., DonselaarWolterink, I., Robbins, T.W. and Everitt, B.J., Cholecystokinin-dopamine interactions within the nucleus accumbens in the control over behaviour by conditioned reinforcement. Behav. Brain Res., 55 (1993) 223±231. Schalling, M., Friberg, K. and Seroogy, K., Analysis of expression of cholecystokinin in dopamine cells in the ventral mecencephalon of several species and human with schizophrenia. Proc. Natl. Acad. Sci. USA, 87 (1990) 8427±8431. Sheng, M. and Greenberg, M.E., The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron., 4 (1990) 477±485. Shilling, P.D., Kelsoe, J.R., Kuczenski, R. and Segal, D.S., Differential regional zif268 mRNA expression in an escalating dose/binge model of amphetamine-induced psychosis. Neuroscience, 96(1) (2000) 83±90. Studler, J.M., Reibaud, M., Herve, D., Blanc, G., Glowinski, J. and Tassin, J.P., Opposite effects of sulfated cholecystokinin on DA-sensitive adenylate cyclase in two areas of the rat nucleus accumbens. Eur. J. Pharmacol., 126 (1986) 125±128. Swerdlow, N.R., Mansbach, R.S., Geyer, M.A., Pulvirenti, L., Koob, G.F. and Braff, D.L., Amphetamine disruption of prepulse inhibition of acoustic startle is reversed by depletion of mesolimbic dopamine. Psychopharmacology (Berl)., 100 (1990) 413±416. Vaccarino, F.J., Nucleus accumbens dopamine-CCK interactions in psychostimulant reward and related behaviors. Neurosci. Biobehav. Rev., 18 (1994) 207±214. Wang, J.Q., Smith, A.J. and McGinty, J.F., A single injection of amphetamine or methamphetamine induces dynamic alterations in c-fos, zif268 and preprodynorphin messenger RNA expression in rat forebrain. Neuroscience, 68 (1995) 83±95. Wolf, M.E., Dahlin, S.L., Hu, X.T., Xue, C.J. and White, K., Effects of lesions of prefrontal cortex, amygdala, or fornix on behavioral sensitization to amphetamine: comparison with N-methyl-D-aspartate antagonists. Neuroscience, 69 (1995) 417±439. Woodruff, G.N., Hill, D.R., Boden, P., Pinnock, R., Singh, L. and Hughes, J., Functional role of brain CCK receptors. Neuropeptides, 19 (1991) 45±56.