Marco Fiore et al- Clozapine or Haloperidol in rats prenatally exposed to methylazoxymethanol, a compound inducing entorhinal-hippocampal deficits, alter brain and blood neurotrophins’

8/3/2019 Marco Fiore et al- Clozapine or Haloperidol in rats prenatally exposed to methylazoxymethanol, a compound induci

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167Ann Ist super sAnIt 2008 | Vol. 44, no. 2: 167-177

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Address or correspondence: Luigi Aloe, Istituto di Neurobiologia e Medicina Molecolare, Sezione NGF, Consiglio Nazionale

delle Ricerche, Via del Fosso di Fiorano 64, 00143 Rome, Italy. E-mail: [email protected].

Smmary. Rats exposed during prenatal lie to methylazoxymethanol (MAM) display in postnatalage structural and behavioral decits resembling those observed in schizophrenic patients. Thesedecits are associated with signicant changes in brain nerve growth actor (NGF) and brain derivedneurotrophic actor (BDNF), particularly in the hippocampus and entorhinal cortex. In the presentstudy, we used the MAM model to investigate in young rats the eect o antipsychotics, Clozapineand Haloperidol, on brain and blood NGF and BDNF presence. Young animals were used becauseadministration o antipsychotics during adolescence is a common eature o intervention. The re-sults showed that administration o Clozapine and Haloperidol causes signicant changes in theconcentration o NGF and BDNF in the brain and bloodstream o MAM-treated rats. These nd-ings indicate that these drugs may aect the synthesis and release o neurotrophins in the centralnervous system and in the blood circulation. In addition, the MAM model can be a useul tool toinvestigate the biochemical and molecular mechanisms regarding the eects o antipsychotics.

Key words: NGF, BDNF, MAM, haloperidol, clozapine, schizophrenia.

Riass(Clozapina o Aloperidolo in ratti esposti in ase prenatale a Metiazossimetanolo, un compo-

sto che induce danni nella corteccia entorinale e nellippocampo, altera le concentrazioni nel sangue enel cervello delle neurotrofne). Il trattamento prenatale con metilazossimetanolo (MAM) determinanel ratto alterazioni morologiche e comportamentali comparabili a ci che si osserva nel pazienteschizorenico. Questo complesso di eventi associato con cambiamenti dei livelli basali di nervegrowth actor (NGF) e di brain derived neurotrophic actor (BDNF) nel cervello del ratto, in parti-colare nellippocampo e nella corteccia entorinale. In questo lavoro abbiamo studiato nel modelloanimale MAM di ratto giovane gli eetti di antipsicotici come la clozapina e laloperidolo sulla pre-senza nel cervello di NGF e BDNF dal momento che questi armaci sono comunemente utilizzatisugli adolescenti. I risultati mostrano che sia la clozapina e laloperidolo modicano i livelli di NGFe BDNF nel sangue e nel cervello e indicano che questi armaci possono ar cambiare la sintesi e ilrilascio delle neurotrone nel sistema nervoso centrale e nel sistema circolatorio. I dati evidenzianoanche che il modello MAM pu essere un approccio utile allo studio degli antipsicotici.

Parole chiave: NGF, BDNF, metilazossimetanolo, aloperidolo, clozapina, schizorenia.

IntRoductIon

Epidemological studies indicate that gestational andperinatal alterations in brain neurogenesis may in-crease the risk o developing behavioral and/or neu-ropathological decits leading to schizophrenia duringearly and late post-natal lie [1]. This neurodevelop-mental disorder is characterized by impaired cognitiveand social perormances, disruption o brain cytoar-chitectural and neural plasticity in the limbic system,

particularly in the entorhinal cortex and hippocampus[2-4]. There is also evidence that maternal starvation,inection, and anoxic birth injury can lead during post-natal lie to behavioral and brain structural decits re-sembling those observed in schizophrenia [5-7].

It has been shown [8, 9] that intererence with neu-rogenesis in the mediotemporal allocortex o the ratembryos, during the earliest stages o cortical proli-eration, results not only in a thickness reduction o

Clozapine or Haloperidol in rats prenatallyexposed to methylazoxymethanol, a compound

inducing entorhinal-hippocampal decits, alterbrain and blood neurotrophins concentrations

Mar Fir(a), Vria di Fas(a), Agla Iailli(b) a Ligi Al(a)

(a)Istituto di Neurobiologia e Medicina Molecolare, Sezione NGF,Consiglio Nazionale delle Ricerche, Rome, Italy

(b)Facolt di Scienze della Formazione, Dipartimento di Scienze e Tecnologie Biomediche,Universit degli Studi, LAquila, Italy


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168 Marco Fiore, Veronica Di Fausto, Angela Iannitelli, et al.

the adult entorhinal cortex and hippocampus [9, 10]but also in other morphological characteristics thatmay be compared with the changes observed in thehippocampal-entorhinal axis o patients with schiz-ophrenia disease [11]. An animal model displaying

these structural brain decits was obtained admin-istering a single injection o methylazoxymethanolacetate (MAM) in pregnant rat on gestational day12 (GD12) or 11 when the entorhinal-hippocampalaxis was supposed to be in major cell prolieration[12]. Indeed in the rat it has been reported that pre-natal MAM administration o [13, 14], at a specictime point o prenatal limbic and cortical develop-ment, causes cell loss and reduces the thickness o thehippocampus, the entorhinal cortex and other brainstructures [8, 15-17]. MAM (reviewed in [13]) is ashort-acting alkylating agent that methylates nucleic

acids leading to the death o cells actively replicatingDNA. In the rat the reaction o MAM with nucleicacids lasts 2-24 h ater injection and is maximal at12 h. As the dierent regions o the brain prolier-ate at overlapping but dierent times, it is possible,

in principle, to produce relatively specic damage,depending on the exact time o drug administration.Prenatal MAM administration induces also loss oneurons, impaired neuroplasticity, cognitive decits,and alterations in the basal levels o neurotrophins[18, 19] and these changes are long lasting since theywere ound at dierent ages (see Table 1).

Nerve growth actor (NGF) and brain-derived neu-rotrophic actor (BDNF) are neurotrophins promot-ing the dierentiation and survival o a number obrain developing neurons. NGF and BDNF contrib-ute to the survival and plasticity o adult neurons in

tabl 1 | Some biochemical and behavioral parameters analyzed ollowing prenatal MAM given in rats at gestational day 11(GD11), 12 (GD12); means increasing, means decreasing, means no changes, NE means not examined

Age Young[51]Youngadult[15]Fullyadult[10,64]Aged[18]

24days 60days 6months >1year

NGF Entorhinal cortex

GD12 GD11 NE

BDNF Entorhinal cortex

GD12 (p=0.08) NE GD11 NE NE

NGF Hippocampus

GD12 (p=0.07) GD11 NE

BDNF Hippocampus

GD12 NE GD11 NE NE

ChAT Meynerts nuclei

GD12 NE NE GD11 NE NE

ChAT SeptumGD12 NE NE GD11 NE NE

Learning performance in the water maze

GD11 NE NE

GD12 NE NE NE Passive avoidance response

GD11 NE NE NEGD12 NE NE NE

TrkA Entorhinal cortex

GD12 NE NE NEGD11 NE NE NE NE

TrkA Hippocampus

GD12 NE NE NEGD11 NE NE NE NE

TrkB Entorhinal cortexGD12 NE NE NEGD11 NE NE NE NE

TrkB HippocampusGD12 NE NE NEGD11 NE NE NE NE


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both normal conditions and ollowing injuries [20-22]. Neurotrophins are known to play also a crucialrole on survival and plasticity o orebrain cholinergicneurons involved in cognitive processes undergoingdegeneration in memory-loss-associated disorders,

including Alzheimer and schizophrenia [23-27].It has been reported that the basal level o NGF

in the bloodstream undergoes signicant changesin patients aected by schizophrenia [28-30], aterHaloperidol administration [31, 32] or electro-con-vulsive therapy [33] and that schizophrenic patientshave low NGF levels [32, 34] or presence o NGFautoantibodies [35]. Moreover, it has been postulat-ed that modulation o the synthesis and secretion oneurotrophins, such as NGF and BDNF by antide-pressants might be a novel approach to treat depres-sion and other brain diseases including Alzheimerand Parkinson [36, 37]. These observations raised

the question as whether a short or acute administra-tion o antipsychotics as Clozapine or Haloperidol,drugs that are widely used in psychiatric disorders[38-40], might aect the synthesis and release oNGF and BDNF in the blood and brain regions inyoung rats since antipsychotics are widely used dur-ing childhood or treating psychiatric disorders. Tourther explore the unctional role o these neuro-trophins, we also investigated the expression o theirmain receptors, TrkA and TrkB respectively, and thepresence o granulocytes in the blood since thesecells are known to be aected by antipsychotics ad-ministration [41].

MethodS

Subject housing and MAM treatment

Gestating Sprague Dawley rats (Rattus norvegicus)were obtained rom an animal arm (Charles River,Italy), which mated animals over a period o 4 h,on the day considered as day 0 o gestation (GD0).Dams with a vaginal plug were separated rom themales and transported to the laboratories. Upon ar-rival at the laboratory, animals were housed in a roomwith air conditioned (temperature 21 1 C, relativehumidity 60 10%), with white lights on rom 7:00

a.m. up to 7:00 p.m. in Plexiglas boxes with a metaltop and sawdust as bedding. Regular rat pellet oodand water were available ad libitum. According toprocedures previously described [15, 42], pregnantrats were randomly divided in the MAM and Salinegroups. Ten pregnant rats or the MAM group un-derwent a single intraperitoneal injection o MAM(22 mg/kg) on gestational day 12 (GD12). This pe-riod o gestation is crucial or the neurogenesis othe hippocampal-entorhinal axis and alterationduring this time may selectively impair cell migra-tion and also may lead to cell death. MAM was pur-chased rom NCI Chemical Carcinogen ReerenceStandard Repository Midwest Research Institute,Kansas City, MO, USA. As controls we used dams(10 rats) which received saline solution (22 mg/kgi.p.) on GD12. At birth animals were ostered to the

biological dams ollowing behavioral procedurespreviously described [42] and used also or otherMAM studies [8, 18]. Litters were also reduced to 3males and 3 emales.

Male animals prenatally administered with MAM

or saline were tested at a month o age. Six groupswere considered: MAM-Cloza, MAM-Halo, MAM-Controls, Sal-Cloza, Sal-Halo and Sal-Controls.The rst 3 groups received MAM in utero, the sec-ond 3 groups received saline in utero. At a month oage MAM and SAL rats were administered daily or8 consecutive days with vehicle, Clozapine (Cloza)or Haloperidol (Halo). MAM-Controls and Sal-Controls (n = 10 or both) were injected i.p. withvehicle (0.8% acetic acid in glucose). MAM-Haloand Sal-Halo (n = 10 or both) received i.p. 2 mg/kgo haloperidol (Janssen-Cilag, Cologno Monzese,Italy). MAM-Cloza and Sal-Cloza (n = 10 or

both) received i.p. 20 mg/kg o clozapine (NovartisPharma, Origgio, Italy). All procedures involvingchemicals preparation, dosages and administra-tion were perormed ollowing methods previouslydescribed [43, 44] involving the eects o antipsy-chotics in the hippocampal region. We used younganimals because the awareness o the onset duringchildhood o psychiatric disorders is rapidly increas-ing. This issues includes administration o antipsy-chotics during adolescence that is at the present timea common method o intervention with times anddosages o administration extremely variables [45].Female rats were used or other experiments.

All eorts were made to minimize and reduce animalsuering and or limiting the number o animal used.All animal experiments were carried out ollowing theprocedure described by the guidelines o the EuropeanCommunity Council Directive o 24 November 1986(86/609/EEC). The Haloperidol has been taken intoaccount since it represents a typical antipsychotic act-ing mainly on D2 receptors. Clozapine has been usedsince it is the rst member o the atypical antipsychot-ics with a wide mechanism o action binding bothdopaminergic and serotoninergic receptors [39]. Thedistinctive binding abilities o the 2 molecules lead todierent clinical applications and to dierent eects

on neurotrophins.Animal sacrifce, and tissue dissection

Rats were killed with a guillotine, the brain quicklyremoved and the hippocampus, entorhinal cortex andstriatum dissected out using a rat brain matrix (ASIInstruments, Inc. Co. USA) [see also the methodol-ogy described by Cuello, 46] and stored at -70 C untilused.

NGF and BDNF determination

NGF/BDNF evaluation (n = 5 or group, one ani-mal per litter) was carried out in the hippocampus,entorhinal cortex and striatum o the rat brain witha ELISA kits NGF Emaxtm ImmunoAssay Systemnumber G7631 and BDNF Emaxtm ImmunoAssaySystem number G6891 by Promega, (Madison, WI,


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USA) ollowing the instructions provided by the man-uacturer. The brain tissues were homogenized withultrasonication in extraction buer (Tris-acetate 20mM, pH 7.5, NaCl 150 mM, EDTA 1mM, EGTA 1mM, Sodium-Pyrophosphate 2.5 mM, Ortovanadate

1 mM, -Glycerolphosphate 1 mM, NaF 100mM,PMSF 1 mM, Leupeptin 1 g/ml) and centriuged at4 C or 10 min, 13 000 rpm and supernatants wererecovered (EDTA, Ethylenediamine-Tetraacetic ac-id; EGTA, Ethyleneglycol-Tetraacetic acid; PMSF,Phenylmethylsulonyl Fluoride). Briefy, 96-well immu-noplates were coated with 100 l per well o polyclonalanti-NGF antibody. Ater an overnight incubation at4 C, the plates were washed one time with wash bu-er (Tris-HCl 20 mM, pH 7.6, NaCl 150 mM, 0.05%Tween 20) and then blocked or 1 hour with block &sample 1x buer provided by manuacturer (200 l orwell). Ater washing the samples were incubated in the

coated wells (100 l each) or 6 h at room temperaturewith shaking. Ater an additional ve washes the im-mobilized antigen was incubated with an anti-NGFmonoclonal antibody overnight at 4 C. The plateswere washed again with wash buer, and then incubat-ed with an anti-rat IgG HRP (Horseradish Peroxydase)conjugate (100 l/well) or 2.5 h at room temperature.Ater washing the plates were incubated with a TMB/Peroxidase substrate solution or 15 min (100 l/well)(TMB, Tetramethyl Benzidine) provided by the manu-acturer. Reaction was then stopped with 100 l/well1N HCl. The colorimetric reaction product was meas-ured at 450 nm using a microplate reader (Dynatech

MR 5000, PBI International, USA). NGF/BDNFconcentrations were determined, rom the regressionline or the NGF/BDNF standard (ranging rom 7.8to 500 pg/ml puried NGF or BDNF) incubated un-der similar conditions in each assay. Under these con-ditions, the recovery o NGF or BDNF in our assayranged rom 80 to 90%. The NGF sensitivity o theassay was about 3 pg/g o wet tissue and cross-reac-tivity with other related neurotrophic actor (BDNF,Neurotrophin-3 and Neurotrophin-4) was less than3%. The BDNF sensitivity o the assay was about 15pg/ml o wet tissue and cross-reactivity with other re-lated neurotrophic actor (NGF, Neurotrophin-3 and

Neurotrophin-4) was less than 3%. Data are represent-ed as pg/g wet tissue and all assays were perormed induplicate.

Western blotting analysis or TrkA, TrkB

The hippocampus, entorhinal cortex and striatum(n = 5 or group, one animal per litter) were homog-enized in sample buer (0.01 M TRIS-HCl buerpH 7.6, containing 0.25 M sucrose, 0.1 M NaCl, 1mM EDTA, and 1 mM PMSF) and centriuged at10 000 g or 10 min at 4 C. The supernatants werethen used or western blotting. Samples (30 g totalprotein) were dissolved with loading buer (0.1 MTRIS-HCl buer (pH 6.8) containing 0.2 M DTT,4% SDS, 20% glycerol, and 0.1% bromophenolblue), separated by 8% SDS-PAGE, and electropho-retically transerred to polyvinylidene fuoride mem-

brane, (PVDF) or 3 h. The membranes were incu-bated or 40 min at room temperature with blockingbuer (10% non-at dry milk, 10 mM TRIS pH 7.5,100 mM NaCl, 0.1% Tween 20). Membranes werewashed three times or 10 min each at room temper-

ature in Tris-buered saline and Tween (TTBS) (10mM TRIS pH 7.5, 100 mM NaCl, 0.1% Tween 20)ollowed by an incubation or 2 h at room tempera-ture with anti-TrkA 1:1000 (provided by Santa Cruz,CA, USA, catalog number 763:sc-118), anti-TrkB1:1000 (provided by BD Biosciences Pharmingen,San Jose CA, USA, catalog number 610101). Asshown in the instructions TrkA antibody does notreact with TrkB or TrkC. Membranes were washedthree times or 10 min each at room temperature inTTBS and incubated or 1 h with horseradish perox-ydase-conjugated anti-rabbit IgG or TrkA or anti-mouse IgG or TrkB (Cell Signalling, Beverly, Mass.,

USA) as secondary antibodies. The blots were devel-oped with enhanced chemiluminescent (ECL) assay(Amersham Bioscience) as chromophore. Similarresults were obtained in 5 independent Westernblot runs. Band densitometry evaluation, expressedas arbitrary units o gray level, o ve dierent gelruns/blot, was made on a Macintosh computer withthe public domain NIH Image program (developedat NIH, Bethesda, MD; available rom: http://rsb.in-o.nih.gov/nih-image/), which determines the OD othe bands with a gray scale thresholding operation.The OD o -actin bands was used as a normaliz-ing actor. The optical density o-actin bands was

used as an internal control or dierence in sampleloading. For each blotting, normalized values wereexpressed as percentage o relative normalized con-trols and used or the statistical analyses.

Biological activity and quantitative evaluationo NGF released in the bloodstream

The rat pheochromocytoma PC-12 cell line wasused in a unctional assay and quantitative evalu-ation o NGF capable to induce neurite outgrowthas previously described [47, 48]. Briefy the serumo control, Halo, and Cloza MAM-treated rats wasdiluted between 1:2 and 1:50 and added to PC-12

cell cultured in RPMI with 5% o etal bovine se-rum and 10% o horse serum. Ater 24 and 48 hin culture, cells bearing neurites were scored. Thequantitative evaluation was determined accordingto the standard NGF dose response curve obtainedby exposing PC-12 cells to dierent concentrationso puried NGF [47].

Blood collection, cell stainingand granulocyte counting

The blood was collected at the moment o animalsacrice. One hundred mg o blood taken rom eachrats was used to make glass smears. Blood smearswere stained with May-Grunwald-Gimsa method,dried, covered with mounting medium and the gran-ulocytes present in 10 dierent elds at magnica-tion 40X were counted and compared.


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Statistical analysis

Data were analyzed by two-way ANOVA consid-ering as eects the prenatal MAM and the drugsexposure. Post-hoc comparisons were perormed us-ing the Tukey-Kramer method and a dierence o

0.05 or less was considered statistically signicant.

ReSuLtS

Eect o prenatal MAM administrationand drug exposure on rat body weight

In Figure 1 is shown that prenatal MAM aectedthe body weight o young rats (the lines with theblack geometrical drawings). ANOVA revealed thatMAMper se decreased the rat body weight [F(1,40)= 15.71; F(7,280) = 19.43, ps < 0.01 or the maineect o prenatal MAM administration and or theinteraction prenatal MAM x repeated measures,

respectively]. ANOVA did not evidence any signi-cant eect o antipsychotics treatment. However,the body weight o both MAM rats and Saline rats(the lines with the triangles) and administered post-natally with vehicle is highly comparable and no sta-tistical dierences were ound.

Eect o Haloperidol and Clozapine on NGF levels

As shown in Figure 2, Haloperidol and Clozapineadministration alters brain NGF concentration. Inthe hippocampus an increase was evidenced by statis-tical analysis [F(1.24) = 39.51, p < 0.01 or the maineect o prenatal MAM] which resulted to be higher

mainly ollowing Haloperidol [F(2.24) = 12.47, p =0.05 in the interaction prenatal MAM x drug expo-

sure; p < 0.05 in post-hocs]. In the entorhinal cor-tex ANOVA showed a signicant increase in NGF inMAM animals [F(1,24) = 150.48; F(2.24) = 5.90; ps< 0.05, main eect o MAM and interaction prena-tal MAM x drug exposure, respectively] with a morepronounced eect ollowing Clozapine administra-tion [F(1.24) = 23.17; p < 0.05 or the eect o drugadministration; p < 0.05 in post-hocs]. In the stria-tum again prenatal MAM induced elevation in NGFvalues [F(1.24) = 6.58, p = 0.02 in the ANOVA or the

160

80

0

Grams

0 1 2 3 4 5 6 7 8

TimeinDays

MAM-ClozaSAL-ClozaMAM-HaloSAL-HaloMAM-ControlsSAL-Controls

RatsBodyWeight

Fig. 1 | Rat body weight in grams in 30-days old rats prenatallyexposed to 20 mg/kg o MAM at gestational day 12 and adminis-tered with Clozapine (Cloza) or Haloperidol (Halo) or 8 days.

2500

1250

0

NGFpg/ml

MAM/Cloza

MAM/Halo

MAM/Controls

MAM/Cloza

SAL/Halo

SAL/Controls

NGFintheHippocampus

4000

2000

0

NGFpg/ml

NGFintheEntorhinalCortex

600

300

0

NGFpg/ml

NGFintheStriatum

Fig. 2 | Mean concentration ( SEM) o NGF in the hippocam-pus, entorhinal cortex and striatum o saline and MAM treat-ed rats receiving Clozapine (Cloza) or Haloperidol (Halo).Asterisks indicate signifcant dierences between groups(* = p < 0.05; ** = p < 0.01).


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eect o prenatal MAM] with signicant post-hocs(p < 0.05) in MAM-Halo vs SAL-Controls.

Figure 3 reports the eect o serum extract onthe expression o neuritis outgrowth rom PC-12cells. The table indicates that prenatal MAM andadministration o Clozapine or Haloperidol causes

a signicant increase in NGF in the serum [F(1.24)= 398.41; F(2.24) = 15.24, ps < 0.05 in the ANOVAor the main eect o both prenatal MAM anddrug exposure, respectively]. The NGF presentin the bloodstream was biologically active, sinceit stimulated the growth o neuritis (arrows) inMAM-Halo rats or MAM-Cloza rats compared toSAL-Controls rats in a classic biological PC-12 cellassay within 48 h. In Figure 3 microphotographsrom MAM-Controls, SAL-Halo and SAL-Clozarats were not included.

Eect o Haloperidol and Clozapineon brain BDNF levels

Figure 4 shows that in the entorhinal cortex statisticalanalysis showed an interaction MAM exposure x drugexposure [F(2.24) = 3.85, p = 0.01] caused by the el-evated levels o BDNF in animals exposed prenatally to

MAM and administered postnatally to Clozapine. In thestriatum MAM exposure in etus increases the levels oBDNF ollowing Haloperidol treatment, while decreas-ing ater Clozapine administration [F(2.24) = 11.35, p

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Marco Fiore et al- Clozapine or Haloperidol in rats prenatally exposed to methylazoxymethanol, a compound inducing entorhinal-hippocampal deficits, alter brain and blood neurotrophins’