BMY 14802, a potential antipsychotic drug, increases expression of proneurotensin mRNA in the rat striatum

Molecular Brain Research, 12 (1992) 279-284

279

1992 Elsevier Science Publishers B.V. All fights reserved. 0169-328X/92/$05.00 BRESM 70379

Research Reports

BMY 14802, a potential antipsychotic drug, increases expression of " proneurotensin mRNA in the rat striatum Beth Levant 1, Kalpana M. Merchant 1, Dan/el M. Dorsa 3-6 and Charles B. Nemeroff 1'2 Departments of 2Pharmacology and 2Psychiatry, Duke University Medical Center, Durham, NC (U.S.A.) and Departments of 3Pharmacology~ 4Medicine, and SPsychiatryand Behavioral Sciences, University of Washington, Seattle, WA (U.S.A.) and Geriatric Research, Education and Clinical Center, VA Medical Center, Seattle, WA (U.S.A.) (Accepted 27 August 1991)

Key words: BMY 14802; Neurotensin; Proneurotensin mRNA; Striatum; Antipsychotic

Treatment with efficacious antipsychotic drugs, such as haloperidol, increases the concentrations of neurotensin (NT) in the nucleus accumbens and caudate nucleus oi the rat. The increases in NT content produced by haloperidol have been shown to be associcated with an increase in NT biosynthesis as assessed by the level of expression of proNT mRHA. The potential antipsychotic drug and o receptor iigand BMY 14802 has been shown to produce increases in the concentrations of NT in the nucleus accumbens and caudate that are similar to those produced by haloperidol with respect to the magnitude of the increases and the time course over which they occur. This study evaluated the effects of BMY 14802 on the expression of proNT mRNA and NT concentration in the rat striatum. Three hours after a single injection of BMY 14802 (35 mg/kg, i.p.) expression of proNT mRNA was significantly increased in the nucleus accumbens and caudate. Labeling was most intense in the dorsal candate. NT concentrations were unaltered at this time point. Eighteen hours after injection, significant increases in NT concentration in the nucleus accumbens and caudate were detected. At this time, expression of proNT mR.NA was substantially reduced compared to 3 h after treatment although labeling of the dorsal and medial candate was still greater than that observed in controls.

INTRODUCTION

A variety of evidence suggests the involvement of the peptide neurotransmitter, neurotensin (NT), in the mechanism of action of antipsychotic drugs and/or the underlying pathophysiology of schizophrenia tS. Briefly, numerous anatomical, neurochemical, electrophysiological, and pharmacological findings describe interactions between the central NT and dopawJne (DA) systems. Of particular interest is the observation that although NT does not alter the binding of D A antagonists to DA receptors, many of the pharmacological effects of centrally administered NT in anhrmls (such as decreased conditioned avoidance responding, antagonism of amphetamine-induced locomotor activity, and induction of hypothermia) are strikingly similar to those prodaced by clinically efficacious antipsychotic drugs. Furthermore, the concentration of NT in CSF of drug-free schizophrenic patients has been repeatedly shown to be decreased when compared to age- and sex-matched controis8'16'27 and has been observed to increase to normal levels after neuroleptic treatment 27. In rats, both acute and chronic treatment with clinically efficacious antipsychotic drugs, such as haloperidol,

chlorpromazine, and pimozide, produce increases in NT concentrations in specific brain regions, most notably the nucleus accumbens and ca!,date nucleus7'1°'13a9, while phenothiazines without antipsychotic efficacy, such as promazine and promethazine, do not t°. These observations led to the hypothesis that NT might represent a type of endogenous neuroleptic-like substance 22. Although alterations in the concentration of a neurotransmitter indicate that it is likely that changes in the neuronal activity of that system are occurring, studies of content alone provide no information on the functional significance of the increase in content or the cellular mechanism(s) by which the increase came about. Recent studies have demonstrated that the increases in NT concentrations observed after haloperidol treatment are likely to result, at least in part, from an increase in NT biosynthesis as assessed by expression of proNT mRNA 2°'2s. Williams et al2s observed an increase in the number of proNT mRNA-containing neurons in the nucleus ~ccumbens and ,,audate nucleus after treatment with haloperidol. Furthermore, the increases in expression of proNT mRNA appears to develop quite rapidly with increased levels of prowl" mRNA in the caudate observed within I h after a single injection of h~ioperidol

Correspondence: C.B. Nemeroff, Department of Psychiatry, Emo~- University School of Medicine, 1701 Uppergate Drive, Atlanta, GA 30322, U . S A .

280 though increases in the nucleus accumbens were not observed in this study2°. BMY 14802, a non-antidopaminergic o receptor ligand, produces behavioral effects in animals, such as antagonism of amfonelic acid-induced hyperactivity and diminished conditioned avoidance responding 26, which have wan'anted its classification as a potential antipsychotic drug. BMY 14802 produces alterations in the concentrations of NT that are similar to those produced by haloperidol with respect to the regional specificity of the brain regions where alterations are observed, the magnitude of the increases in NT concentration, and the time course over which the alterations occur ~. The present study sought to evaluate the effects of BMY 14802 on NT biosynthesis in the rat striatum as assessed by the steady state level of proNT mRNA and the immunoreactive concentrations of NT. MATERIALS AND METHODS

Experimentof procedure Adult, male, Sprague-Dawley rats (150-175 g; Charles River Labs, Raleigh, NC) were housed 2 per cage with flee access to laboratory chow and water. The temperature- and humiditycontrolled animal facility had a 12 h dark-light cycle. Rats were obtained 1 week before the commencement of each experiment and were accustomed to" handling. Rats were treated with a single injection (i.p.) of BMY 14802 (Bristol-Myers Co., Wallingford, CT) at a dose of 35 mg/kg, administered in 0.3% tartaric acid vehicle in a volume of 2 ml/kg. Control animals were treated with 0.3% tartaric acid. Rats were sacrificed by decapitation 3 h or 18 h after injection. All animals were sacrificed between 10.00 and 11.00 h. For measurement of proNT mRNA, brains (n -- 5) were frozen in isopentane and stored at -70"C until sectioning. For measurement of NT concentrations, brains (n -- 7) were frozen on dry ice and stored at -70°C until dissection.

In situ hybridization assay for proneurotensin mRNA Rat proNT mRNA was detected using a 3ss-labeled 39 base synthetic oligonucleotide probe complementary to bases 171-209 of the rat cDNA sequence (5'dGAGCTCTCTrGGTGCTTCCTCTTGCC[TCAGCTGGCTFG). End-labeling of the oligonucleotide was accomplished using [35S]dATP and terminal deoxynucleotidyl transferase (TDT) according to methods previously described4. DTT was added to the probe fraction to a concentration of 10 mM. The specificity of this probe for NT mRNA has been established on the basis of the following criteria: (a) complete lack of hybridization signal in plesence of 100.fold excess of unlabeled probe, (b) the antomical patterns of hybridization signal generated by the 5'directed oligonucieotide used in the present study were identical to those obtained by hybidization with a 3'-directed riboprobet, and (c) examination of emulsion-coated sections under dark- and lightfield microscopy illustrated that the silver grains representing hybridization were distributed specifically over cell bodies in all regions examine¢. Saturation of hybridization with this probe occurs between I and 2 pmol/ml of hybridization mix. In ~itu hybridization was carried out by a modification of methods previously describedI. Serial coronal brain sections (20 #m) were thaw-mounted onto gelatin-coated slides and stored at -70°C ~tntil processing for hybridization. For processing, slides were win:ned to room temperature for I0 rain, fixed in 4% paraformaldehyde, acetylated with 0.25% acetic anhydride (in 0. I M triethanolamine, pH 8), dehydrated through a graded alcohol series, delipidated in chloroform, and rehydrated in 95% ethanol prior to air-drying. RNAse-

free procedures were used throughout. The probe (2 pmol/ml) was applied in 45 /~1 of hybridization buffer contaiug 0.3 M salt and 50% formamide in 10 mM Tris buffer (pH 8.0) and spread over sections by the application of silanized coverslips. Slides were incubated at 30"C (Tm-20*C) in a moist chamber overnight. After removal of the cove[slips in 1 x SSC (0.15 M .~aCl, 0.015 M sodium citrate, pH 7.0), slides were washed (4 × 15 rain) in 1 × SSC at 55°C (T,,-15*C). Two additional washes were performed at room temperature in 1 x SSC. Sections were then dehydrated through a graded alcohol series in which water was replaced by 0.6 M ammonium acetate and air-dried. Slides were apposed to Hyperfilm-~max (Amersham) for 3 weeks. Films were developed in D19 solution (Kodak). Brain regions were identified according to Paxinos and WatsonzS. Labeling was considered specific if it was bilaterally symetrical, and consistent from section to section and brain to brain. For quantification, autoradingrains were digitized with an Amersham-Loats RAS-1000 image analysis system. Film back-ground was subtracted from each image. Average pixel optical density (O.D.) was determined for each brain region sampled. The image analysis system had a resolution of 512 x 512 pixels. Pixei size was calibrated to 0.04 mm2. Brain regions were defined by the atlas of Paxinos and Watsonzs. O.D.s were determined at three levels through the nucleus accumbens (bregma 2.2 mm, 1.7 mm, and 1.2 mm). The caudate was subdivided into dorsal, medial, and ventral subregions (Fig. 3). In this brain region, O.D.s were determined at four levels (bregma 1.2 mm, 0.4 mm, -0.4 mm, and -1.3 mm). Globus pallidus was also sampled at two levels (bregma -0.4 and -1.3). Slides ~,'ere subsequently coated with Kodak NTB2 emulsion (diluted 1:1 with 0.6 M ammonium acetate), exposed for 3 weeks, developed in D19 solution (diluted 1:1 with H20) and counterstained with Cresyl violet acetate for microscopic examination.

Neurotensin radioimmunoassay For measurement of NT concentrations, the nucleus accumbens, anterior caudate, posterior candate, and substantia nigra/ventral tegmental area were isolated by freehand dissection on ice by a modification of the Glowinski and Iversen method9. Brain tissues were extracted by sonic dismembranation in ice cold ! N HCI in polypropylene microfuge tubes. Homogenates were centrifuged at 8000 g for 15 min at 4°C; supernatants transferred to new polypropylene microcentrifuge tubes. Duplicate aliquots of supernatant were transferred to borosilicate glass tubes on ice. The aliquots were lyophilized in a Savant concentrator, reconstituted in assay buffer, and assayed by equilibrium radioimmunoassay according to methods previously described in detail2. The antiserum is directed towards the midportion of the ~ molecule, amino acids 6, 7 and 8 (Lys-Pro-Arg) and was used at a final dilution of 1:14,000. Synthetic NT(1 -13) was used as standard and was iodinated for use as a tracer. Goat anti-rabbit antiserum (Arnel Products, New York) was used as the second antibody. The assay has a sensitivity of 1.25 pg/tube and an ICso of 90 pg/tube. Recovery or"NT from both tissue homogenate and extract is approximately 99%. Interassay variability is approximately 20% at a concentration of 50 pg/tube. Intraassay variability is approximately 10%. Pellets resulting from l~F extraction were dissolved in 1 N NaOH and assayed for protein concentration by the method of Lowry et al. 17. Bovine serum albumin (BSA) is used as standard in the protein assay.

Statistics Quantitative data from autoradiograms are expressed as O.D./ region and as the mean ± standard error of the mean (S.E.M.). Data were analyzed by two-way analysis of variance (ANOVA) with factors of brain subregion and time after administration. Rostro-caudal variation within brain regions was analyzed by twoway ANOVA with factors of rostro-caudal level within the brain region and time after administration. Because no rostro-candal differences were detected, the mean O.D. over all levels of a given brain region was used for further analyses. For brain regions where a significant treatment effect was detected, ANOVA was followed

281 pothalamic nuclei, the nucleus accumbens, bed nucleus of the stria terminalis, and preoptic area. Labeling of the Control caudate was relatively sparse in keeping with the relatively low density of immunoreactive N T cell bodies in this brain region 6. Three hours after injection of BMY 14802, a large in:rease in labeling of the caudate was observed (Fig. 1). Although labeling of the entire brain region was increased, labeling was most L~tense in the dorsal portion of the caudate (Fig. 2). An increasv in the expression of proNT m R N A was also observed in the nucleus accumbens, though it was somewhat smaller in magnitude than that observed in the caudate. Statistically significant rostro-caudal gradients were not observed in either brain

by the Student-Newman-Keuls :nultiple comparisons test. Radioimmunoassay data are expressed as pg NT/mg protein. Data for all treatment groups are expressed as the mean ± S.E.M. Data were analyzed for statistical significance by two-way ANOVA with factors of brain region and time after administration. Data were log-transformed for thJ'~procedure because of inhomogeneity of variance. Where siguific~,ntinteractions were observed, one-way ANOVA was performed for each brain region. A significant oneway ANOVA was followed by the Student-Newman-Keuls muigi~" pie c~mparisons test. RESULTS Control animals exhibited a distribution of proNT mRNA similar to that reported by Alexander et al,1 (Fig. 1). Moderate to dense labeling occurred in certain hy-

¸:'¸,7

CONTROL

!

BHY 14802 - 3h

BI4Y 14802 - 1 8 h

Fig. 1. Effects of BMY 14802 on expression of proneurotensin mRNA in the striatum. Rats (n = 5 per group) were sacrificed 3 h and 18 h after treatment with BMY 14802 (35 mg/kg, i.p.). Autoradiograms with film backgound subtracted are shown in rostro-caudal sequence (bregma 2.2 mm, 0.7 mm, -0.3 mm, -1.3 mm). CPu, caudate-putamen, NA, nulceus accombens; GP, giobus pallidus.

~2 TABLE I

20-

Effects of BMY 14802 on regional neurotensin concentrations

Values are mean -+ S.E.M. Rats (n = 7 per group) were sacririced 3 h and 18 h after injection with BMY 14802 (35 mg/kg, i.p.). Brain region

Neurotensin (pglmg protein) Time after injection.

Nucleus accumbeus Anterior caudate Posterior candate Substantia nigra/ventral tegmental area

Control

3 hours

18 hours

403 +- 18 84 -+ 5 160 + 17

406 - 14 81 -+ 5 151 -+ 22

501 -+ 21"* 138 -+ 8** 242 -+ 24*

765 - 51

753.4- 81

799 --- 49

**P < 0.01, *P < 0.05 when compared to control by ANOVA and the Student-Newman-Keuls multiple comparisons test.

region. Levels of proNT m R N A were unaltered in the globus pallidus. Increased labeling was also observed in the septal area while labeling was decreased in the hypothalamus (data not shown). No alterations in N T concentrations were yet detected at this time point (Table I). Eighteen hours after treatment with BMY 14802, significant increases in the concentrations of N T were observed in the nucleus accumbens and anterior and posterior caudate (Table I). However, labeling of prowl' mRNA in these brain regions was substantially reduced at 18 h compared to labeling observed 3 h after treatment. Even so, labeling of the dorsal and medial caudate was still intensified relative to control (Fig. 2).

0

15.

i10

!, 0

I'--I CONTROL IS~ 3h r'x'l 18h

**tt

**tt

,.li1 NUCLEUS ACCUMBE]t5

,t

MNDII~.

CAUDATE

GLOBUS PN..UDUS

Fig. 2. Quantification of the effects of BMY 14802 on regional levels of proneurotensin mRNA. Rats (n ffi 5 per group) were sacrificed 3 h and 18 h after treatment with BMY 14802 (35 mg/kg, i.p.). Autoradiograms were quantified with a RAS-1000 image analysis system. Division of the caudate nucleus is shown in Fig. 3. Data is expressed as average optical density (O.D.) ×100 per region sampled. **P < 0.01, *P < 0.05 when compared to control, tip < 0.01, *P < 0.05 when compared to 18 h by ANOVA and the Student-Newman-Keuls multiple comparisons test.

DISCUSSION The present study clearly demonstrates that the increase in the concentrations of N T in the nucleus accumbens and caudate observed after treatment with BMY 14802 is preceded by an increase in the steady-state level of the m R N A encoding the p r o N T peptide within perikarya located in these brain regions. The present study did not investigate whether the increase in p r o N T m R N A content was a result of an increase in N T gene transcription or an increase in the stability of the m R N A . However, it is likely that the rate of N T biosynthesis was increased as a result of the increase in its m R N A content following treatment with B M V 14802. The delay between the detection of increased level of proNT m R N A

Fig. 3. Division of the caudate nucl~m for analysls of autoradk~v,~, To facilitate quantification of optical densities, the caudate nucleus was subdivided into dorsal (D), medial (M), and ventral (V) subre~ons.

283 and increased NT content likely represents the time period required for translation and post-translational processing of the prohormone into h~l". The effects of BMY 14802 on the expression of proNT mRNA are similar to those produced by treatment with haloperidol2°'2s. Differences exist, however, in the anatomical localization of the most intense labeling of the caudate and the detection of increased mRNA in the nucleus accumbens. Thus the haloperidol-induced increase in hybidization signal was most significant in the dorsolateral region of the caudate2°. In contrast BMY 14802 increased the expression of NT mRNA in both the dorsolaterai and the dorsomedial aspects of the neostriatum. These differences may represent differential effects of the two drugs or differing experimental designs. The absence of a detectable increase in proNT mRNA in the nucleus accumbens observed by Merchant et al.2o may be related to the early time point at which levels of message were determined or the low dose of haloperidol employed (0.5 mg/kg). Time course studies with BMY 14802 indicate that increased NT concentration in the caudate is detectable earlier than in the nucleus accumhens14. This may also be true for haloperidol. The dose of BMY 14802 employed in the present study produces similar increases in NT concentrations to that observed after haloperidol (1 mg/kg), intermediate between the doses of Merchant et al. 2° and Williams et al. 2s (2 injections at 2 mg/kg, 7 h interval). The distribution of increased labeling of proNT mRNA after treatment with BMY 14802 is also concordant with the localization of increased NT-immunoteactive perikarya in the nucleus accumbens and caudate, particularly the dorsomedial caudate, after treatment with 2 doses of haloperidols. In the present study, the location of intense labeling following treatment with BMY 14802, which indicates the location of NT cell bodies, does not coincide with the normal (untreated) distribution of NT-immunoreactive cell bodies in this brain region -- the few NT cell bodies being concentrated in the ventral and medial areas6. It therefore appears that the observed increases in the level of proNT mRNA anse from synthesis by cells that produce less-than-detectable levels of immunoreactive NT in control rats -- a mechanism that would clearly facilitate an increase in NT neurotransmission. The previously described decrease in the activities of peptidases which degrade NT after treatment with antipsychotic drugs21 might also facilitate an increase in neurotransmission. Central administration of NT has been shown to produce a variety of effects that are strikingly similar to those produced by antipsychotic drugs including the antagonism of certain DA-mediated behaviors. Accordingly, an increase in NT neurotransmission represents a mecha-

nism by which the effects of DA neuronal activity could be antagonized without the blockade of DA receptors. Previous investigation of the effects of BMY 14802 and haloperidol on regional brain NT concentrations suggested that the drug produced its effects on the NT system through int¢ractions with a non-dopaminergic receptor, most likely the putative o r e c e p t o r t4. The localization of increased labeling of proNT mRNA after treatment with BMY 14802, however, provides only limited information concerning the receptor interactions responsible for the increases in NT concentrations. Although levels of proNT mRNA were increased throughout the nucleus accumbens and caudate, the increase in labeling was particularly dramatic in the dorsal caudate. Labeling appeared to be non-striosomai or unrelated to the location of striosomes. Thus, the neurons expressing increased NT biosynthesis appear to be proximal to neurons positive for acetylcholinesterase (ACHE). In the caudate, AChE has been associated with both acetylcholine and DA neurons24. DA nerve terminals, positive for tyrosine hydroxylase, are present in high density throughout the nucleus accumbens and caudate but in much lower d¢,mity in the giobus pallidus TM. Both D1 and D 2 DA receptors are present in high density thoughout the nucleus accumbens, caudate, and globus pallidus23. Although present in lower density than DA receptors, a receptors have also been shown to be fairly uniformly distributed throught these brain regions3'11'1s. The distribution of increased proNT mRNA after treatment with haloperidol or BMY 14802 most closely approximates the distribution of NT immunoreactive fibers in the striatum. The distribution of tyrosine hydroxylase-positive DA neurons is also similar to the distribution of proNT mRNA. This is not suprising considering the well documented interactions between the HT and DA systems. In summary, the present data demonstrate that the increases in the NT concentrations of nucleus accumbens and caudate produced by BMY 14802 are preceded by an increase in the levels of mRNA encoding proNT peptide. These BMY 14802-induced increases in ~mmunoreactive NT content and proNT mRNA are not unlike those produced by haloperidol. While it is tempting to speculate that BMY 14802 and haloperiol may alter concentrations of NT and proNT mRNA via a connnon mechanism, further studies will be required to deten~ine how these drugs influence the NT system. Acl~owledgcmen~. We are grateful to Dr. Duncan Taylor of Bristol-Myers Co. for generouslysupplyingthe BMY 14802. Supported by NIMH MH-39415 (B.L., C.B.N.), the Department of Veterans Affairs Research Service, NS20311 (D.M.D.), and the Washington Institute for Mental Illness Research and Training (K.M.M., D.M.D.).

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