Kalpana M. Merchant, Paul R. Dobner and Daniel M. Dorsa- Differential Effects of Haloperidol and Clozapine on Neurotensin Gene Transcription in Rat Neostriatum

8/3/2019 Kalpana M. Merchant, Paul R. Dobner and Daniel M. Dorsa- Differential Effects of Haloperidol and Clozapine on Ne

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The Journal of Neuroscience, February 1992, 72(2): 652-663

Differential Effects of Haloperidol and Clozapine on NeurotensinGene Transcription in Rat NeostriatumKalpana M. Merchant, Paul R. Dobner,4 and Daniel M. Dorsa1.2.3Department of Pharmacology and 2Departments of Medicine and Psychiatry & Behavioral Sciences, University ofWashington, Seattle, Washington 98195, 3Geriatric Research, Education and Clinical Center, Seattle VA Medical Center,Seattle, Washington 98108, and 4Department of Molecular Genetics and Microbiology, University of Massachusetts,Worcester, Massachusetts 01655

A single dose of typical neuroleptic, haloperidol, has beendemonstrated to increase the expression of neurotensin/neuromedin N (NT/N) mRNA in the dorsolateral striatum with-in 1 hr of its administration (Merchant et al., 1991). Thepresent study further investigated neuroleptic-induced reg-ulation of NT/N gene transcription. Levels of NT/N mRNAwere examined at various times following a single dose ofhaloperidol(1 mg/kg, i.p.) or the atypical antipsychotic clo-zapine (20, 30, or 40 mg/kg, i.p.) by in situ hybridizationhistochemistry and quantitative solution hybridization. In thedorsolateral striatum, the two drugs had strikingly differenteffects; haloperidol rapidly (within 30 min) increased the ex-pression of mature NT/N mRNA while virtually no increasewas observed in response to nontoxic doses of clozapineat any of the time points examined. Following haloperidol,maximal induction occurred at 7 hr, at which time NT/N ml?NAlevels were an order o f magnitude higher than basal levels.By 20 hr after haloperidol, there was a significant decline instriatal NT/N mRNA levels. In situ hybridization analysis us-ing an intron-derived probe revealed that haloperidol-in-duced increases in mature NT/N mRNA levels in the striatumwere preceded by a transient increase in intron-containingNT/N gene transcripts. These data strongly indicate that acutehaloperidol treatment results in transient transcriptional ac-tivation of NT/N gene, although a concomitant eff ect on thestabili ty of NT/N primary transcripts cannot be ruled out. Incontrast to their differential eff ects in the dorsolateral stria-turn, a single dose of both haloperidol and clozapine induceda small but significant increase in NT/N mRNA expressionin the shell sector of the nucleus accumbens. These resultsraise the possibility that NT neurons in the nucleus accum-bens may, at least in part, mediate the antipsychot ic eff ectsof classical neuroleptics, whereas NT cells in the dorsolat-

Received May 6, 1991; revised Aug. 28, 1991; accepted Sept. 27, 1991.We are grateful to Dr. Ariel Deutch for his guidance in anatomical definitionof nucleus a ccumbens core and shell. This research was supported by grants fromWashington Institute for Mental Illness Research and Training (K.M.M. andD.M.D.), by Sco ttish Rite Schizop hrenia Research Program wM.M. and D.M.D.),bv Resea rch Service Deoartment ofveterans Affairs fD.M.D.). and bv NIH GrantsNS 20311 (D.M.D.) and HL 33307 (P.R.D.). We thank Anne Kall~omaki, Kim-berley Donnell, and Cheryl Refsdal for their excellent technical assistance. Weare also grateful for the generous gift of clozapine by Sandoz Pharmaceuticals.Correspondence should be addressed to Kalpana M. Merchant, Ph.D., 182-B,GRECC,.Seattle VA Medical Center, 1660 South Columbian Way, Seattle, WA98108.

Copyright 0 1992 Society for Neuro scienc e 0270-6474/92/12065 2-12$05.00/O

era1 region of the striatum may be involved in mediating othereffect s of typical neuroleptics such as extrapyramidal motorsymptoms.Neurotensin (NT) is a tridecapeptide originally isolated frombovine hypothalamus (Carraway and Leeman, 1973)and is het-erogeneously istributed in the CNS, where t is likely to func-tion asa classical eurotransmitter or neuromodulator (Uhl andSnyder, 1976; Kitabgi et al., 1977; Iversen et al., 1978; Youngand Kuhar, 1981; Uhl, 1982).A variety ofrecent studies ndicatethat central NT pathways may play an important role in theetiology and/or pharmacotherapy of schizophrenia and otheraffective mental disorders. For example, anatomical and bio-chemical evidence indicates that NT modulatesdopaminergicpathways (for reviews, seeQuirion, 1983; Emson et al., 1985;Levant et al., 1990) mplicated in the etiology of schizophrenia(Seeman,1987). Additionally, drug-free schizophrenicpatientshave significantly lower concentrations of NT in their cerebro-spinal fluid ascompared o their age-and sex-matchedcontrols,and upon treatment with antipsychotic drugsNT concentrationreturns to normal in these patients (Widerlov et al., 1982b).Furthermore, the biochemical (e.g., ncrease n dopamine um-over) (Widerlov et al., 1982a; Kalivas et al., 1983) as well asbehavioral responsese.g., decreased onditioned avoidance e-sponse,decrease n amphetamine-induced ocomotion, hypo-thermia) (Bissetteet al., 1976; Ervin et al., 1981) to centrallyadministeredNT are reminiscentof the effectsof clinically usedneuroleptic drugs. In fact, these observations have led to thesuggestionhat NT may be an endogenous euroleptic-like com-pound (Nemeroff, 1980).Neuroleptic drugsare a group ofchem-ically diverse compounds showingan excellent correlation be-tween heir dopamineD, receptor-blocking efficacy and potencyfor antipsychotic effects Creese t al., 1976;Seeman t al., 1976).However, NT neither binds to the D, receptors nor directlymodulatesCAMP accumulation causedby dopamine receptoragonists Nemeroff et al., 1983).Hence, the cellular mechanismunderlying the neuroleptic-like effectsof NT remainsunknown.Several studies have demonstrated that administration ofneuroleptic drugs ncreases he concentration of immunoreac-tive NT in striatal regionsof the rat (Govoni et al., 1980; Freyet al., 1986; Letter et al., 1987; Eggermanand Zahm, 1988).Recently, we have shown that the increase n striatal peptidecontent following an acute singledoseof haloperidol is accom-panied by a dramatic increase n the neurotensin/neuromedinN (NT/N) mRNA levels (Merchant et al., 1991).Hence, halo-


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The Journal of Neuroscience, February 1992, 12(2) 653

peridol-induced increases in the synthesis and possibly releaseof the endogenous neuroleptic NT could underlie some of thepharmacological effects of this classical antipsychotic. Haloper-ido l is a prototype of what have been termed typical antipsy-chotic drugs, which are known to have a high propensity toinduce extrapyramidal motor side effects (EPS) in patients. In-terestingly, in our initial study (Merchant et al., 199 1) we foundthat the effects ofa single acute dose of haloperidol were confinedprimarily to the dorsolateral region of the striatum in the rat.This region is a part of the basal ganglia circuitry implicated inregulation of motor output and is not thought to be a part ofthe limbic systems involved in mediating antipsychotic effects.Therefore, it raises the possibility that the haloperidol-sensitiveNT neurons in the motor striatal region may be involved inmediating some of the acute EPS (e.g., dystonia, parkinsonism)caused by this drug. If true, the atypical antipsychotic, clo-zapine, which is relatively free of acute EPS (Gerlach et al.,1975) would not be expected to influence NT/N gene expressionin these neurons.In the present study, we have compared and characterizedthe acute effects of haloperidol and clozapine on NT/N mRNAexpression using the techniques of in situ hybridization histo-chemistry and solution hybridization in order to understand hefunctions of the dorsolateralneostriatal NT cells.Additionally,we have studied the molecular mechanismunderlying the in-crease n levels of NT/N mRNA causedby haloperidol. Anincrease n the content of a mature mRNA species ould resultfrom either increased ranscription or posttranscriptional reg-ulation suchas an increase n mRNA stability or transport outof the nucleus Guyette et al., 1979; Hynes et al., 1979; Mc-Knight and Palmiter, 1979). Using an antisenseRNA probederived from an intervening sequence n the NT/N gene, wehave examined the effects of haloperidol on NT/N nuclear pri-mary transcripts asan index of changesn the rate of transcrip-tion of the gene. This approach has been used successfully ostudy pro-opiomelanocortin gene transcription in rat brain (Fre-meau et al., 1989).Our results demonstrate that the expression of NT/N mRNAin a subsetof neurons ocated in the dorsolateral region of theneostriatum is differentially regulated by prototypes of typicaland atypical neuroleptics (haloperidol and clozapine, respec-tively). On the other hand, NT/N mRNA expression n theaccumbalshellwasenhancedsimilarly by these wo drugs.Thisraises he possibility that anatomically distinct populations ofNT neurons may be involved in mediating motor side effectsand antipsychotic effects of clinically used neuroleptic drugs.Additionally, the effectsof haloperidol on NT/N mRNA appearto be primarily a nuclearevent involving synthesisand/or tum-over of NT/N primary transcripts.

kg; McNeil Pharmaceuticals), clozapine (20, 30, or 40 m&g; SandozPharmaceuticals), or vehicle (1 ml/kg). At various times after treatment,rats were killed by decapitation between 12:00 noon and 3:00 P.M.Brains were rapidly removed, frozen on dry ice, divided sag&ally intotwo halves, and stored at -80C until processed for in situ hybridizationor quantitative solution hybridization assays.Synthesis of probes. The probe used for detection of mature NT/NmRNA was synthesized in vitro using the method of Melton et al. (1984)from a 336 base pair EcoRV/Bgl II fragment of NT cDNA (nucleotides626-96 1) subcloned into BamHVSmaI-digested DGEM~ (Promeaa). Thespecifici ty o f this subclone (prNT4) has been- established p&ously(Alexander et al., 1989; Merchant et al., 199 1). EcoRI-linearized prNT4was used as a template for the antisense RNA probe labeled with eitherYS-UTP (0.9-l x lo8 dpm/pmol) for in situ hybridization or with 32P-UTP (4-6 x 10 dpm/pmol) for solution hybridization. A sense RNAtranscript was synthesized from the Hind III-linearized prNT4 withtrace amounts of 3J-I-UTP for generating a standard curve in the solutionhybridization assay as described below.-To generate anintron-specific subclone (pNTgHX), a unique-se-quence 1 O kilobase HindIII/XmnI fragment derived f rom intron 2 ofthe rat NT/N gene was inserted into HindIII/SmaI-digested pGEM3ZF( -) (Promega). Antisense intron probe for in situ hybridization wastranscribed f rom HindIII-linearized plasmid using T7 RNA polymeraseand labeled to a specific act ivi ty of 3-5 x lo* dpm/pmol with Yj-UTP.A sense probe was also synthesized to the same specif ic activ ity fromEcoRI-linearized olasmid usina SP6 RNA oolvmerase. Radiolabelednucleotides and enzymes were obtained from-New England Nuclear andBoehringer Mannheim, respectively.In situ hybridization histochemistry. A minor modification of previ-ously described methods (Alexander et al., 1989; Merchant et al., 199 1)was used. Brief ly, sagitta lly halved brains were cut into 20-pm-thickslices, thaw mounted onto gelatin-coated slides, and stored at -80Cuntil orocessed as follows. The slides were warmed to room temnerature(RT)for 10min, fixed in 4% w/v paraformaldehyde, acetylated with0.25% v/ v acetic anhydride in 0.1 M triethanolamine (pH 8.0) dehy-drated through a graded series of ethanol, delipidated in chloroform,rehydrated to 95% v/ v ethanol, and air dried. Adjacent sections wereused to hybridize with either the coding region- or the intron-specificprobes described above. The labeled probe was applied at a saturatingconcentration (1.5-2 pmol/ml) in a hybridization solution [ 10 mM Tris-HCl buffer, pH 8.0, containing 50% v/v deionized formamide, 0.3 MNaCl, 1 mM EDTA, 10%w/v dextransulfate, x Denhardts olution(Sambrook et al., 1989), 10 mM dithiothreitol, and 0.5 mg/ml yeasttRNA]. Sections ere overedwith siliconized overslips,ndhe slideswere ncubated or 16-18 hr in a humidchamber t 20Cbelow hecalculated eltingemperature53Cor thecodingegion-specificrobeand 50C for the intron-specific probe). The coverslips were removedin 1 x SSC (0.15 M NaCl + 0.015 M sodium citrate, pH 7.0), and theslides were washed in 1 x SSC for 30 min at RT. This low-strinaencvwash was followed by RNase treatment 120 &ml RNase A in a buffercontaining 10 mM T&-HCl (pH 7.4), 0.5 M EDTA and 0.5 M NaCl] at37C for 3w5 min. The slides were then rinsed in the buf fer used forRNase treatment at 37C for 30 min followed by three high-stringencywashes (20 mineach)n 0.1x SSC t 15C below the theoreticalmeltingtemperature (52C for the coding sequence-specific probe and 50C forthe intron-specific probe). The slides were subsequently dehydratedthrough a graded alcohol series in which water was substi tuted by 0.6M ammonium acetate and air dried.

Materials and MethodsAnimals and drug treatment. Adult male Sprague+Dawley rats (200-250 gm; Simonsen Laboratories, Gilroy, CA) were housed two to threeper cage in a temperature-controlled environment with 12 hr light/l2hr dark cycle and were given free access to standard laboratory chowand water. In a pilot study, the effects of stress caused by handling and/or an intraperitoneal injection were assessed by comparing the expres-sion of NT/N mRNA in four separate groups of animals (n = 4): in-jection naive but handled 30 min prior to death or treated with saline(1 ml/kg, i.p.) at 30 min, 1 hr, or 3 hr prior to death. None o f thesegroups differed in the distribution of NT/N mRNA-containing cells.To study the effectsof neuroleptics on NT/N mRNA expression, animalswere treated with a single intraperitoneal injection of haloperidol(1 mg/

To determine the specific ity of the intron-specific probe, some sec-tions were treated as described above except labeled sense probe o f thesame specif ic act ivi ty was substituted for labeled antisense probe.Autorudiogruphy. For film autoradiography following hybridizationwith the coding region probe, slides were apposed to Hyperfilm-@max(Amersham) for 2-3 d and films were developed in Kodak D-19 so-lution. Slides were then dipped in Kodak NTB2 nuclear tract emulsiondiluted 1: 1 with 0.6 M ammonium acetate, air dried in the dark for 2hr, and exposed for either 6 d (for the coding region probe) or 15 d (forthe intron-specific probe). The emulsion was developed in D- 19 diluted1: 1 with water. Sections were counterstained in 0.1% w/v cresyl violetacetate, dehydrated, and coverslipped with Permount. Brain sectionsfrom different groups were anatomically matched according to the atlasof Paxinos and Watson (1986) using bright-field microscopy prior toexamination of the distribution of autoradiographic grains in dark field.Quantification of hybridization signal from film autoradiograms wascarried out by a person blind to the experimental design. Autoradio-


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654 Merchant et al. * Effe cts of Antipsycho tics on Neurotensin Gene Transc ription

IEgure 1. Film autoradiograms showing the time course of haloperi-dol-induced increase in NT/N mRNA-expressing cells. Animals (n =6) were given a single dose of haloperidol(1 mg/kg, i.p.) and killed at0.5, 1, 3, 7,20, or 48 hr following treatment. The distribution of NT/NmRNA-expressing cells was studied using 20 pm sections from thesag&tally halved brain of each animal by in situhybridization with aYS-labeled antisense RNA probe (prNT4) derived from the coding re-gion of the NT/N gene. Film autoradiograms were generated by apposingsections to Hyperfd-Bmax for 48 hr. Sections at a single level of thestriatum (bregma 1 mm) are shown. Arrowsndicate dorsolateral stria-turn (DLS), the area most affected by haloperidol treatment. C, control;h,hr.

grams were digitized with a Drexels Unix-based Microcomputer ImageAnalysis System. Background optical density was subtracted from eachimage. Optical densities in the shell region of the nucleus accumbens(see Fig. 5) were determined at a single level (bregma 2.2 mm) fromfour different sections per animal. For the dorsolateral striatum, theoptical densities were determined using four sections from each animalat the coronal plane 1 mm anterior from the bregma.Solutionhybridization.This technique has recently been adapted inour laboratory from the methods of Paul et al. (1988) and is found todetect small amounts (0.5 pg/tube) of NT/N mRNA satis factorily intissue extracts. Brains were cut into 300-pm-thick slices. The dorsolat-era1 striatum and the nucleus accumbens (encompassing both core andshell) were microdissected on a cold plate maintained at - 10C usingthe atlas of Palkovits and Brownstein (1988). The dorsolateral neostri-atum was excised using a knife from atlas levels: A3000 pm to A300pm. Striata from two to three animals were pooled for each assay. Thenucleus accumbens was dissected using a 200 pm punch, and due tosmall amounts of the tissue, punches from all animals were pooled.Tissues were homogenized and total RNA was extracted as describedpreviously (Chomczynski and Sacchi, 1987). Sense (with H tracer) andantisense (with 3ZP abel) RNA transcripts from the prNT4 subclonewere synthesized and purified as described above. A standard curve wasgenerated by hybridizing increasing amounts (0.5-200 pg) of the sensetranscript with a fixed amount (50.pg; 20,000-30,000 dpm) of the an-tisense urobe in hvbridization buffer (0.3 M NaCl. 4 mM EDTA. 406forma&de, 0.2 mg/ml yeast tRNA, and 20 mM Tris-HCl, pH 7.4). Twodifferent concentrations of the total RNA extracted from the tissueswere hybridized with the same amount of antisense RNA probe at 60Cfor 16-18 hr. Following hybridization, samples were mixed with 1 mlof RNase buffe r containing 25 pg/ml RNase A and 500 U/ml RNaseT 1 and digested for 90 min at 37C. Nuclease digestion was terminatedby addition of 100 pl of 100% chilled trichloroacetic acid. Samples wereincubated on ice for 20 min, filtered through prewetted nitrocellulosefilters, dried, and counted in scintillation fluid. The amount of NT/NmRNA per mg of total RNA for each sample was calculated using thestandard curve.Statistics. lterations in NT/N mRNA concentrations in the dorso-lateral stria tum and the nucleus accumbens were quantified by solutionhybridization or densitometric analysis of the film autoradiograms asdescribed above. Data are presented as mean f SEM. Differences be-tween means were analyzed using ANOVA. Following a significant dif-ference in the variance, Schef fes test was applied to identify groupsdiffering signif icantly from control values. Differences were consideredsignificant if the probability that they were zero was less than 5%.

ResultsCharacterization of acute effects of haloperidol on matureNT/N mRNA. Expression of NT/N mRNA was examined atvarious times following the administration of a single dose ofhaloperidol(1 mg/kg, i.p.) using a coding region antisense RNAprobe for in situ hybridization. As can be seen in Figure 1,haloperidol increased NT/N mRNA expression predominantlyin the dorsolateral region of the neostriatum within 30 min (theearliest time point examined). Maximal expression of NT/NmRNA was observed between 3 and 7 hr after drug adminis-tration, followed by a significant decline at 20 hr, and the ex-pression returned to control levels by 48 hr following haloperidoltreatment. Although the largest increases were observed in thedorsolateral striatum, small increases in NT/N mRNA-express-ing cells were detected in the nucleus accumbens, particularlyin the shell sector of the nucleus accumbens (Figs. 2,3) at 3 and7 hr after treatment. These increases were apparent even thoughthe basal expression of NT/N mRNA in the nucleus accumbensin control rats was much higher than that in the caudate-pu-tamen. The increased expression of NT/N gene in the dorso-lateral striatum or the nucleus accumbens was not an ef fec t ofinjection-related stress as determined in a pilot study (see ani-mals and drug treatment under Materials and Methods). Sig-nificant hybridization to NT/N mRNA in control rats was also


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The Journal of Neuro science , February 1992, 72(2) 655

Figure 2. Distribution of NT/N mRNA-containing cells at the peak of the haloperidol effect. Film autoradiograms from the study described inFigure 1 are shown at three different rostral-caudal levels of the striatum. A (bregma 2.2 mm), B (bregma 0.7 mm), and C (bregma -0.8 mm) arerepresentative sections from saline-treated animals, whereas D, E, and F are anatomical ly matched typical sections from animals killed at 7 hrfollowing haloperidol. Arrows ndicate hybridization signal in dorsolateral striatum (DLS), nucleus accumhens (M), olfactory tubercle (OT), andpreoptic region (PO). S, Septal nuclei; GP, globus pallidus.

observed in several other areas such as the septal nuclei, olfac-tory tubercle, piriform cortex, and the preoptic region of thehypothalamus. However, haloperidol did not appear to alterNT/N mRNA levels in these regions (Fig. 2).To quantitate the haloperidol-induced increases in NT/NmRNA levels in the dorsolateral striatum, this region was mi-crodissected from the contralateral brain of animals used forthe in situ hybridization study described above and the contentof NT/N mRNA was determined by quantitative solution hy-bridization. In concordance with the in situ assay, NT/N mRNAlevels were increased time dependently by haloperidol (Fig. 4).The maximal increases were observed at 7 hr following drugadministration, at which time NT/N mRNA content in thedorsolateral striatum was approximately an order of magnitudehigher than the control levels. Haloperidol effects on NT/NmRNA expression in the shell of nucleus accumbens were quan-tified by densitometric analysis of the film autoradiograms. Hy-bridization to NT/N mRNA in the nucleus accumbens shellsignificantly increased at 3 and 7 hr following haloperidol (Fig.54) and subsequently declined to control levels by 20 hr after

drug treatment. The haloperidol effect in the nucleus accumbenswas also evident in a 45% increase in NT/N mRNA contentdetermined by solution hybridization using a pool of punchesencompassing both the shel l and the core of the nucleus accum-bens dissected from the contralateral hemisected brains of sameanimals (Fig. 5B).Efects of clozapine on mature NT/N mRNA expression. Un-like ha loperidol, a single dose of clozapine (20 mg/kg, i.p.) didnot appear to affect the number of NT/N mRNA-expressingcells significantly in the dorsolateral striatum at 1 hr, 3 hr, or 7hr following treatment (Fig. 6). Examination of emulsion-coatedslides under dark field revealed a few hybridization-positive cellsin the caudate-putamen at 1 and 3 hr after treatment. However,these cells were not tightly localized in the dorsolateral regionas seen with haloperidol but were scattered unevenly in thedorsal striatum. Thus, following a single administration of 20mg/kg of clozapine, the amount of NT/N mRNA in three sep-arate pools of total RNA from dorsolateral caudate did notincrease over control leve l at any of the time points examined(Fig. 4). A subsequent dose-response study revealed that even


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656 Merchant et al. l Effe cts of Antipsycho tics on Neurotensin Gene Transc ription

Figure 3. Effects of haloperidol and clozapine on NT/N mRNA expressionn the nucleus accumbens. atswere reatedwith a single ose fhaloperidol(1 mg/kg), clozapine (20 mgkg), or vehic le (1 ml/kg). In situ hybridization was carried out using the coding region probe as describedin Figure 1. Autoradiograms were generated after 6-7 d exposure to Kodak NTB2 emulsion. B-D are low-magnification, dark-field photomicrographsthrough the nucleus accumbens and represent the region of the brain schematically shown in A by the shaded rectangle.he photomicrographsrepresent typical hybridization in a control section (B),or from rats treated with haloperidol for 7 hr (C) or clozapine for 3 hr (0). Clusters of silvergrains epresent ellsexpressing T/N mRNA. * Indicates nteriorcommisure.

at 30 mg/kg (ip.), clozapine remained ineffective in inducingNT/N mRNA expression n the dorsolateral striatum (Fig. 7).However, at 40 mg/kg, an apparently toxic dose at which oneout of five animals n the group died, a significant increase nhybridization signal n the dorsolateral striatum was observed.In contrast to the caudate-putamen,3 hr following 20 mg/kgof clozapine, a significantly higher expressionof NT/N mRNAwas observed in the nucleus accumbens Figs. 3, 6). As withhaloperidol, this effect waspredominantly observed n the shellsector. Quantitative analysis of film autoradiograms ndicateda significant ncreasen the optical density in the accumbal shellfollowing clozapine (Fig. 54). Solution hybridization using otalRNA pooled from the entire nucleus accumbens eflected thisclozapine-induced increase n NT/N mRNA content (Fig. W),which wascomparable n magnitude o that observedwith halo-peridol.Haloperidol increases nuclear levels of intron-containingNT/Nprimary transcripts.An antisenseRNA transcribed froma nonrepetitive, intronic sequence of NT genomic DNA(pNTgHX; Fig. 8) was used to study the distribution of cellscontaining NT/N primary transcripts following treatment withsaline or haloperidol. The specificity of this probe for nuclearprimary transcripts was tested by comparing the cellular distri-

bution of autoradiographic grains generatedby this probe withthose produced by hybridization to the coding region probe.The grains ollowing hybridization with the intron-specific probeshowedcompact localization predominantly over the nuclei ofneurons, whereas hose generatedby the coding region probewere scattered around the cytoplasmic portion of the hybrid-ization-positive cells (Fig. 9). Additionally, no specific hybrid-ization was observed when a senseRNA probe synthesized othe samespecific activity was employed (Fig. 9C). In situ hy-bridization with the antisense ntron sequenceprobe to brainsectionsadjacent to those used o study the distribution of ma-ture NT/N mRNA did not show any specific hybridization inthe dorsolateral striatum of saline-treated ats (Fig. 1OA). At 30min following haloperidol, an increase n the number of cellsexpressing ntron-containing NT/N transcripts was evident inthe dorsolateral caudate (Fig. 10B). A further increase n hy-bridization to the nuclear transcripts wasobserved by 1 hr afterhaloperidol (Fig. lOc), but by 3 hr, hybridization-positive cellswere no longer apparent in this region (Fig. 1OD). No hybrid-ization was observed at subsequent ime points (7, 20, 48 hr)in the dorsolateral caudate-putamen data not shown). Follow-ing haloperidol, cells at their peak expression of intron-con-taining NT/N transcripts (detected by the intron probe) ap-


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The Journal of Neuroscience, February 1992, V(2) 667

time (hr) following time (hr) followinghaloperidol clozapine

Figure 4. Quantification of the eff ects of antipsychotic drug treatmenton dorsolateral striatal NT/N mRNA expression. Dorsolateral striatumwas microdissected from the contralateral brains of the animals usedfor in situ hybridization analysis . Levels of NT/N mRNA were deter-mined by solution hybridization using a 32P-labeled, antisense codingregion probe (prNT4). Each bar represents mean NT mRNA contentf SEM in two (haloperidol) or three (clozapine) separate pools of totalRNA. * P


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656 Merchant et al. * Effe cts of Antipsycho tics on Neurotensin Gene Transc ription

Figure 6. Film autoradiogramshow-ing the effectsof clozapineon distri-butionof NT/N mRNA-expressingellsin thestriatum.Rats n = 6)were reat-ed with a singlenjectionof clozapine(20mg/kg, .p.) or vehicleandkilledat1, 3, or 7 hr after he treatment. n situhybridization was carried out as de-scribed n Figure 1. Autoradiogramsrepresent 2 hr of exposureo Hyper-film-Bmax.Typical sectionsreshownat the sameevel bregma mm)of thestriatum. C, Control; CZ, clozapine.Double arrows point at the hybridiza-tion signaln the dorsolateral triatum(DLS), andasingle arrow indicates y-bridization n the nucleus ccumbensWA).

between he potency of antipsychotic drugs o induce catalepsyin rats and acute production of EPS in humans. Hence, it istempting to speculate hat the response f discreteNT neuronsin the caudate-putamen to antipsychotics may be used as ascreeningassay o predict their liability to induce acute EPS(such as dystonia, parkinsonism) n humans. In this regard, itis important to note that cholecystokinin and enkephalin pep-tides that also closely interact with central dopamine systemsare not differentially affected by typical and atypical antipsy-chotics (Frey, 1983;Angulo et al., 1990).However, further stud-ieswith a variety of typical and atypical antipsychotic drugsarerequired n order to understand he potential role of various NTneuronal populations in mediating specific pharmacologicalef-fects of thesedrugs.In contrast to the caudate nucleus,NT/N mRNA expressionin the nucleus accumbensshell was ncreased ollowing treat-ment with both haloperidol and clozapine, although the mag-nitude of this increasewas much smaller han that observed nthe dorsolateral caudate (Figs. 3, 5). Additionally, haloperidol-induced increases n the nucleus accumbensoccurred after alonger lag time than in the caudate-putamen Figs. 4, 5). Thesedata are consistent with the observation that increases n im-munoreactive NT content of the nucleusaccumbens ausedbyhaloperidol occur later and are of smaller magnitude han thoseoccurring in the caudate-putamen (Frey et al., 1986; Letter etal., 1987; Merchant et al., 1988a). The longer lag time alsoexplains why we previously failed to observe a significant in-crease n NT/N mRNA content in the nucleusaccumbens1 hrafter haloperidol treatment (Merchant et al., 1991). In contrastto the present study, Williams et al. (1990) observed largerincreasesn NT/N mRNA expression n the nucleusaccumbensand the ventral striatum following two dosesof haloperidol(2mg/kg, i.p.) 17and 10hr prior to death. Additionally, this dosing

regimen did not appear o increaseNT/N mRNA expression nthe dorsolateralstriatum. Thus, distinct populations of NT neu-rons appear to be differentially regulated in response o singleor multiple dosesof haloperidol and/or the recovery time al-lowed following drug treatment. Supporting this, we have ob-served that at 18 hr following three dosesof haloperidol (6 hrintervals) there is a selective increase n the number of NT/NmRNA-expressing cells in the nucleus accumbensand ventralcaudate-putamen but not in the dorsal striatum (K. M. Mer-chant, D. M. Dorsa, unpublished observations). It is likely thatmaximal induction in NT/N mRNA in the ventral striatum(including the nucleusaccumbens) equiresmultiple stimuli and/or a longer lag time.Both haloperidol and clozapine causedsimilar increasesnNT/N mRNA expression n the nucleus accumbens,predomi-nantly in the shell sector. Recent anatomical and biochemicaldata indicate that whereas he core of the nucleus accumbensmay be associatedwith the nigrostriatal dopamine system, heshell may be related to the mesolimbic system (Zahm, 1989;Deutch and Cameron, 1991; Heimer et al., 1991). Thus, in-creasedexpressionof NT/N gene n the limbic structure by thetwo prototypes of typical and atypical antipsychotic drugs aisesthe possibility that the shell NT neurons may representa path-way involved in someof the common pharmacological effectsof these wo classes f drugs. Whether it involves manifestationof antipsychotic effects n humans remains to be determined.The selective ncrease n NT/N mRNA content in the limbicstriatum induced by clozapine is consistentwith its preferentialeffects on mesolimbic rather than mesostriatal dopamine sys-tems (Chiodo and Bunney, 1983; White and Wang, 1983). Ad-ditionally, following chronic treatment with clozapine, an in-creasen immunoreactive NT content is observed n the nucleusaccumbens but not in the dorsolateral striatum (Kilts et al.,


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The Journa l of Neu roscienc e, February 1992, 72(2) 659

A

q control* N clozapine-20

Figure7. Dose-response study for the effects of clozapine on NT/NmRNA expression in the dorsolateral striatum. Rats were treated in-traperitonially with a single dose of 20, 30, or 40 mg/kg o f clozapine 1hr prior to death. In situhybridization was carried out as described inFigure 1. A, Film autoradiograms at a single level through the striatum(bregma 1 mm) are shown from a control rat (c) or animals treatedwith various doses ofclozapine (numbers represent the dose ofclozapinein mgkg, i.p.). Arrow ndicates hybridization signal in the dorsolateralstriatum (DLS). B, Quantification of hybridization signal in the dor-solateral striatum was carried out by densitometric analysis. Each barrepresents mean optical density + SEM (n = 5, except in clozapine-40group, where n = 4). *, P < 0.02 when compared to all other groups.

1988). The precise cellular mechanism underlying clozapinesselectivity for the mesolimbic systems s not clear. It is possiblethat clozapines ability to block receptorsother than dopamineD, (e.g., muscarinic, LYEoradrenergic, 5-HT,) renders t moreselective for the limbic pathways compared to the mesostriatalpathways. The responses f striatal NT neurons to concurrentadministration of haloperidol and the antimuscarinic drug tri-hexyphenidyl, for example, would therefore be interesting es-pecially in view ofthe clinical observation that coadministrationof this anticholinergic drug with typical neuroleptics educes heincidenceof EPS (Fann and Lake, 1976;McEvoy, 1983).Recentidentification and cloning of a novel dopamine receptor, termedDq, which appears o be more sensitive to clozapine than D,receptors (Van To1 et al., 199 ), also raises he possibility thatthe selectivity of clozapine may be attributed to its preferential

Intron 2 Exon 3

SP 6 (sense)

pNTgHX

IT 7 (antisense)

Figure8. Schematic diagram of the subcloning strategy used to gen-erate the intron-specific probe. Plasmid sequences (-), intron se-quences (-), and exon sequences (rectangle with diagonal lines) areshown.blockade of D, receptors. Further characterization of the dis-tribution of D, receptors will help identify specificbrain struc-tures targeted by this atypical antipsychotic.The projection fields of the distinct populations of striatal NTneurons remain conjectural. It is likely that the dorsolateralstriatal NT neurons project to the globus pallidus and/or sub-stantia n&a, the two major structures hat receive striatal motoroutput. Gerfen et al. (1990) have recently demonstrated hat D,receptors appear to be functionally associated with striatal neu-rons that project to the globuspallidus whereasD, receptorsareprimarily associatedwith striatal-nigral pathways. In view ofthis, it is likely that the haloperidol-sensitive NT neurons n thedorsolateral striatum project to the globuspallidus. Supportingthis, an increase n NT-immunoreactive fibers and terminals sobserved in the globuspallidus following treatment with halo-peridol (Eggermanand Zahm, 1988). Studies are underway tomap the projection fields of striatal NT neurons. Such studiesshould advance further our understanding of the functions ofthesedistinct NT neuronal populations.In order to understand the molecular mechanismunderlyinghaloperidol induction of NT/N mRNA, the effects of this drugon NT/N primary transcripts were examined. The use of anintron-specific probe for in situ hybridization analysis evealedthat the levels of intron-containing NT/N gene ranscripts wererapidly, but transiently, elevated in the dorsolateral striatumafter a single dose of haloperidol (Fig. 10). The transient ac-cumulation of intron-containing transcripts ndicates that halo-peridol treatment results n the activation of NT/N gene ran-scription, althoughpossible ffectson stability of NT/N precursorRNA cannot be ruled out at present. The anatomical distri-bution of cells abeled by the intron and exon probeswas den-tical (Fig. 11). Additionally, maximal induction of the intron-containing transcripts (observed at 1 hr; Fig. 10) preceded hemaximal induction in the mature mRNA (observed at 7 hr;Figs. 1,4). Thesedata suggesthat the rapid, transient activationof NT/N gene ranscription was responsible or the subsequent


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660 Merchant et al. l Effe cts of Antipsycho tics on Neurotensin Gene Transc ription

Figure 9. Specificity f the ntron-derived robe pNTgHX) for NT/Ntranscripts.High-magnification,right-fieldphotomicrographsf au-toradiogramshrough he dorsolateral triatumof haloperidol-treatedanimals reshownollowingn situ hybridizationwith eitherYS-labeled,antisenseoding-regionprNT4) probe A) or )%-labeled, ntisensen-tron-specificPNTgI-IX) probe B). Dark grainsover stained euronsindicatehybridization-positive ellsarrows). Figure8C s a low-mag-nification,dark-fieldphotomicrographhrough he same egionof thestriatum ollowingn situ hybridizationwith labeled enserobe ran-scribedrom pNTgHX. Autoradiograms eregeneratedy coating hebrain sections ith Kodak NTB2 emulsion nd developing ftera 6 d(coding egionprobe) r 15 d (intron-derivedprobes) xposure.accumulation of mature NT/N mRNA in the dorsolateral stria-turn. The specificity of the intron-specific probe for NT primarytranscripts was also evident from the distinct nuclear localiza-tion of hybridization signaland the failure of a senseRNA probe

to show any hybridization (Fig. 9). The rapid decline in levelsof intron 2-containing transcripts at 3 hr suggestshat the intronsequences re rapidly degradedafter splicing. It was nterestingthat the dorsolateral striatal neurons showed minimal expres-sion of NT/N mRNA in the basal state (Fig. 2). It appears,therefore, that NT/N geneexpression n certain populations ofcentral neurons may depend entirely on appropriate environ-mental stimuli.The transcriptional effects of haloperidol could be mediatedthrough the CAMP pathway and the induction of immediateearly genessuch as c-fos as proposed n Figure 12. BlockageofD, receptors has been shown to increase ntracellular levels ofCAMP and also cause ransient activation of c-fos expression(Miller, 1990). Transient transfection analysis in PC12 cells,which neuronally differentiate in responseo NGF, has evealedthat AP-1 and CAMP-response element (CRE) sequences rerequired for the integration of transcriptional responses f theNT/N gene o multiple environmental stimuli (Kislauskis andDobner, 1990). A family of genes, typified by the c-fos andc-jun proto-oncogenes,encode ranscriptional factors that bindthe AP-1 site with high affinity in vitro (Bartel et al., 1989). Adistinct but related family of proteins bind the CRE with highaffinity (Habener, 1990). A CRE-binding protein identified inPC12 cells, CREB, binds constitutively to the CRE sequences,but its ability to activate transcription is strikingly increasedfollowing phosphorylation by CAMP-dependent protein kinase(Gonzalez and Montminy, 1989). Haloperidol treatment couldresult in the activation of NT/N gene transcription throughincreasedphosphorylation of a CRE-binding protein and thetransient activation of AP- l-binding factors such as c-fos. Thespecific implication of D, receptors in directly mediating thetranscriptional effects of haloperidol on NT/N geneexpressionrequires further study. However, removal of tonic D, receptoractivity hasbeen mplicated in the haloperidol-induced ncreasein striatal NT content (Merchant et al., 1989). A potential roleof CAMP in regulation of NT/N gene transcription is also sup-ported by the observation that activation of D, receptors (pos-itively linked to adenylate cyclase) ncreasesNT content whileD, receptor activation (negatively linked to adenylate cyclase)decreases T content in the striatum (Merchant et al., 1988b).Thus, it appears ikely that haloperidol influences NT/N genetranscription, in part, by altering intracellular CAMP levels, pos-sibly via blockade of D, receptors.Basedon the data presentedhere, an increase n NT biosyn-thesis n the caudate-putamenappears o occur following treat-ment with haloperidol. However, Bean et al. (1989) have re-ported that reserpine-induced ncreases n NT content in theneostriatum may be due to blockade of NT release ather thanan increase n its biosynthesis. Since the effect of reset-pine nNT content appears o be due to removal of D, receptor tone(Merchant et al., 1989), the data presentedhere appear to beinconsistent with the observations of Bean et al. (1989). How-ever, it is likely that NT terminals originating outside he stria-turn may be regulated differently by reserpine as compared tothe cell bodiespresent within the striatum. Reset-pinemay, forexample, decrease he release rom terminals of extrinsic neu-rons and yet increase he synthesis of the peptide in discreteintrinsic neurons n the striatum, the net effect of which will bean increase n the peptide content. On the other hand, subpop-ulations of NT neurons originating in the striatum may be reg-ulated distinctly by dopaminergic blockers. The technique of insitu hybridization histochemistry used n the presentstudy offers


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The Journal of Neuroscience, February 1992, 12(2) 661

the advantage of excellent anatomical resolution to identify dis-crete NT systems that may be differentially regulated.In summary, our results ind icate that both haloperidol andclozapine increase the expression of NT/N mRNA and hencepossibly the biosynthesis of this peptide in the neostriatum.However, there are significant anatomica l differences in the neu-rons targeted by these two drugs, suggesting that a funct ionaldiversity may exist among subpopulations of striatal NT neu-rons. Future studies with a variety of antipsychotic drugs canbe used to determine whether specific NT neuronal populationsin the striatum represent distinct substrates of typical and atyp-ical antipsychotics, thereby resulting in the unique pharmaco-log ical and behavioral profiles of these two classes of drugs.

Figure II. Comparison of anatomical localization of hybridizationsignal generated by coding region and intron-derived probes. Low-mag-nification, dark-field photomicrographs represent autoradiogramsthrough the dorsolateral striatum generated in the studies described inFigures 1 and 9. A and B represent typical autoradiograms from halo-peridol-treated animals at 1 and 7 hr, respectively, following hybrid-ization with the intron-specific probe (A) or coding region probe (II).cc, Corpus collosum.

Figure 10. Dark-field photomicro-graphs showing the time course of halo-peridol induction of intron-containingNT/N transcripts. Brain sections ad-jacent to those used in the study de-scribed in Figure 1 were hybridized with35S-labeled, antisense intron-specificprobe (pNTgHX). Autoradiograms weregenerated by 15 d exposure to KodakNTB2 emulsion. Clusters of silver grainsin the high-magnification, dark-fieldphotomicrographs indicate NT/N pri-mary transcript-containing cells in thedorsolateral striatum from a controlbrain (4) or from haloperidol-treatedrats at 0.5 hr (E), 1 hr (C), or 3 hr (0)following treatment.

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ATP CAMPF V CREBPK-A -L

(fos,un

NT/N gene J

Figure 12. Proposed theoretical model for dopamine D, receptor me-diated regulation of NT/N gene transcription. See text for discussion.Gi, inhibitory G protein; AC, adenylate cyclase.


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662 Merchant et al. l Effects of Antipsychotics on Neurotensin Gene Transcription

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Kalpana M. Merchant, Paul R. Dobner and Daniel M. Dorsa- Differential Effects of Haloperidol and Clozapine on Neurotensin Gene Transcription in Rat Neostriatum